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Marine Biological Laboratory Library 

Woods Hole, Mass. 


Presented by 

John Wiley and Sons Inc» 
New York City 






















General Editors: sir rudolph peters, f.r.s. 
and F. G. YOUNG, f.r.s. 


The Biochemistry 
of Semen 


M.D., Sc.D., Ph.D., F.R.S. 

Reader in Physiology of Animal Reproduction, 
in the University of Cambridge 



First published in 1954 


Printed and Bound in Great Britain by Butler & Tanner Ltd., Fiome and London 

^"^-^ y^ 

LIBS #. 


When I took up my studies on semen in 1944, on behalf of the 
Agricultural Research Council, I became painfully aware of the 
fact that information on the physiology of semen, its chemical 
aspects in particular, is rather difficult to come by; the older 
observations and records being hidden away in books and 
journals not readily accessible in any but the best equipped 
libraries, and moreover, scattered throughout an exceptionally 
wide range of publications, which embrace disciplines as far 
apart as say, agriculture, urology and cytology. Judging from 
numerous requests for information, received from fellow 
workers in the field, biochemists, clinicians, zoologists and 
veterinary officers alike, the absence of a fairly comprehensive 
and up-to-date treatise on the chemical physiology of semen 
must have proved a serious handicap to many in their scientific 
and practical pursuits. Therefore, I accepted gladly the invita- 
tion to write this book; having agreed to produce but a 'little 
book', I have often found it rather irksome to condense the 
vast mass of data into the allotted space; had it not been for 
the encouragement and ready help of colleagues — my wife not 
least among them, the task would have been even more 

Biochemistry of semen is a relatively modern, but rapidly 
expanding, field of physiology; consequently, many of our 
present views, particularly as regards the biological significance 
of various chemical constituents of semen, may have to be 
revised or modified in the near future. That being so, I like to 
look upon this book, or at any rate, those parts of it which 
deal with the newer, still fluid concepts, as something in the 
nature of an Interim Report, designed to furnish information 
and to convey ideas emerging from the state of knowledge as 
available at the time of writing, however imperfect that may be. 
In presenting the recently acquired evidence, I have tried to 
render justice to developments in the sphere of mammalian as 

vi The Biochemistry of Semen 

well as non-mammalian physiology, selecting examples from 
species as far apart as man and the sea-urchins, and occasion- 
ally, introducing plants as well. I have done my best to dis- 
tinguish between established fact and tentative hypothesis, and, 
as far as possible, have refrained from the tendency, currently 
prevalent among workers in this field, to assign to every newly 
discovered chemical constituent of semen a major role in the 
process of fertilization. 

I wish to acknowledge gratefully the help of those who gave 
me permission to reproduce plates and figures. In particular 1 
wish to extend my thanks to Dr. C. R. Austin (Sydney), Dr. 
J. L. Hancock (Cambridge) and the Cambridge University 
Press for Plate I, to Prof. L. H. Bretschneider and Dr. Woutera 
van Iterson (Utrecht) and the Nederland Academy of Science 
for Plate II, to the Royal Society for Plate III, to Lord Roth- 
schild (Cambridge) for Plate IV and for reading the manuscript, 
to the Royal Society of Edinburgh for Fig. 2, to Dr. E. Blom 
(Copenhagen) and the Skandinavisk Veterinartidskrift for Fig. 3, 
to Dr. C. Huggins (Chicago) and the Harvey Society of New 
York for Fig. 5, to Dr. L. Jacobsson (Goteborg) and the Acta 
Physiologica Scandinavica for Fig. 11, and to the Cambridge 
University Press, Messrs. Churchill and Messrs. Macmillan for 
permission to reproduce Figs. 6-10, 12-14 and 16, from the 
Biochemical Journal, the Journal of Agricultural Science, and 
Nature, and Plate IV, from theCiba Foundation Symposium on 
Mammalian Germ Cells. I should also like to thank Miss P. A. 
Northrop for helping me in the preparation of the typescript. 







Spermatozoa. Spermatogenesis and sperm 'ripening'. Sperm 
transport in the female reproductive tract and 'capacitation'. 
Structural and chemical characteristics of the sperm-head, 
middle-piece and tail. 

Seminal plasma. Secretory function of male accessory 
glands. Prostatic secretion. Seminal vesicle secretion. Physio- 
logical significance of seminal plasma. Prostaglandin, vesi- 
glandin, and certain other pharmacodynamically active sub- 
stances. Coagulation and liquefaction. 



Species and individual variations in the composition of 
semen. Pre-sperm, sperm-containing, and post-sperm fractions 
in the ejaculate. Criteria for the rating of semen quality. 
Optical and electrical properties of semen. Viscosity, specific 
gravity, osmotic pressure, and ionic equilibrium. Hydrogen 
ion concentration and buffering capacity. Metabolism of 
semen and its relation to sperm density and motility; glycolysis; 
methylene-blue reduction test; respiration. 



Sperm inhibitors and spermicidal substances. Chemical 
aspects of short-wave radiation. Variations in hydrogen ion 
concentration and tonicity. Influence of heat and cold; sperm 
vitrification and 'la vie latente'. Role of hormones. Sperm-egg 
interacting substances and chemotaxis. 'Dilution effect' and 
chemical changes associated with senescence. The use of 
artificial diluents in the storage of semen. 



viii The Biochemistry of Semen 



Mechanical separation of sperm from seminal plasma; 
release of intracellular proteins from damaged spermatozoa. 
Removal of the sperm nucleus from the cytoplasm. Protein- 
bound iron, zinc, and copper. Cytochrome. Catalase. Hyal- 
uronidase and other 'lytic' agents. Sperm nucleoproteins. 
Deoxyribonucleic acid. The basic nuclear proteins; protamines 
and histones. The non-basic nuclear proteins; karyogen and 
chromosomin. Keratin-like protein of the sperm membrane. 


PLASMA 1 1 1 

Proteoses and free amino acids. Fibrinolysin and fibrino- 
genase. Pepsinogen. Ammonia formation. Amino acid oxidase. 
Seminal phosphatases; 'acid' and 'alkaline' phosphatase; 
5-nucleotidase; pyrophosphatase. Enzymic hydrolysis of 
adenosine triphosphate. 


Lipids in spermatozoa. The lipid capsule. Acetal phospho- 
lipids or plasmalogens. Role of lipids in sperm metabolism. 
Lipids in the seminal plasma and male accessory gland 
secretions. 'Lipid bodies' and prostatic calculi. 


Fructose as a normal constituent of semen. Species differ- 
ences. Site of formation. Seminal fructose as an indicator of 
male sex hormone activity; the 'fructose test' and its applica- 
tion to certain problems of sex endocrinology. Role of hypo- 
physis. The relationship between blood glucose and seminal 
fructose. Effect of malnutrition. The enzymic mechanism of 
fructose formation. Anaerobic and aerobic utilization of 
carbohydrate by spermatozoa. Pasteur effect and the 'meta- 
bolic regulator'. Intermediary reactions in sperm fructolysis 
and the role of phosphorus-containing coenzymes. 



Spermine. Occurrence of crystalline spermine in human 
semen; its chemical nature and properties. Derivatives of 
spermine and their use in forensic medicine. Synthesis of 

Contents ix 


spermine. Spermidine. Oxidation of spermine and spermidine 
by diamine oxidase. State of spermine in semen. 

Choline. The Florence reaction in semen. Enzymic libera- 
tion of choline from precursors in semen. Phosphorylcholine 
and glycerylphosphorylcholine. Physiological function of free 
and bound choUne. Choline esterase. 

Ergothioneine. Isolation of ergothioneine from the boar 
seminal vesicle secretion. The function of seminal ergothio- 
neine and its behaviour towards sulphydryl-binding sub- 
stances. Biogenesis of ergothioneine. 

Creatine and creatinine. Occurrence in mammalian semen 
and in the sperm and gonads of invertebrates. Phosphocreatine 
and phosphoarginine. 

Adrenaline and noradrenaline. Occurrence in semen and 
accessory organs. Enzymic oxidation. Pharmacodynamic 


Citric acid. Occurrence and distribution. Influence of male 

sex hormone. Citric acid in the female prostate. Metabolism 

and role of seminal citric acid. 
Inositol. Occurrence and distribution, m^^olnositol as a 

major constituent of the seminal vesicle secretion in the boar. 

Physiological function. Relation to other seminal constituents. 



INDEX 223 



between pages 1 and 1 1 

facing page 14 










1 Diagrammatic representation of a spermatozoon 4 

2 'Ripening' process in the epididymis of the mouse 7 

3 Schematic representation of the head of a bull spermato- 

zoon before and after detachment of the galea capitis 13 

4 Diagrammatic outline of male accessory organs to illustrate 

the localization of fructose 16 

5 Diagram of osmotically active substances in prostatic 

fluid 18 

6 Relation between volume of ejaculate and content of 

fructose and citric acid in rabbit semen 31 

7 Composition of boar semen fractions collected by the 

'split-ejaculate method' at half-a-minute intervals 37 

8 Fructolysis in bull semen incubated at 37° 47 

9 Effect of fluoride on the respiration and aerobic fructolysis 

of ram semen 51 

10 Effect of fructose, glucose and lactate on the respiration 

of washed ram spermatozoa 53 

11 Increase of non-protein nitrogen and amino-nitrogen 

content in human semen on incubation at 37° 112 

12 Post-castrate fall and testosterone-induced rise of seminal 

fructose in rabbit 140 

13 Dosage-response curves of testosterone propionate, using 

the coagulating glands of the rat 141 

14 Effect of alloxan diabetes and insulin on seminal fructose 

in rabbit 147 

15 Diagrammatic representation of fructolysis in semen 156 

16 Effect of ergothioneine on boar spermatozoa 177 



Before I decided to embark upon the business of studying the 
metabolism of semen, my interest used to centre on very different 
biochemical problems; earlier on, in the laboratory of J. K. Parnas, 
I was youthfully grappling with the intricacies of intermediary 
carbohydrate metabolism in muscle, blood and yeast; later on, at 
the Molteno Institute, in happy association with D. Keilin, we were 
investigating the nature and function of metalloprotein enzymes in 
plant and animal tissues. When confronted with the opportunity of 
an extensive study of spermatozoa, I did not hesitate to give up my 
former pursuits in order to devote myself to experiments involving 
biological material which offers the investigator a chance, almost 
unique so far as mammalian tissues are concerned, of correlating 
chemical and metabolic findings with clearly defined and highly 
specific criteria of physiological activity, such as the motility and 
fertilizing capacity of the spermatozoa. Among other peculiarities 
which make semen such a fascinating and attractive object of study 
is that it represents an animal tissue with but a single type of cells, 
the spermatozoa, freely suspended in a fluid medium of some com- 
plexity, the seminal plasma, and not subject to cellular growth, 
division or multiplication; thus, making it feasible to express all 
one's metabolic measurements directly in terms of cell numbers, 
without recourse to cumbersome and often unreliable standards such 
as dry weight of tissue, nitrogen content, or indeed, any other of the 
commonly used metabolic indices. From the purely practical point 
of view, which matters greatly, the ability of spermatozoa to 'survive', 
i.e. retain their remarkable properties under conditions of long-term 
storage in vitro, is of great importance. This in turn, gives one a 
chance of exploring at will and under well-defined conditions in 
vitro, the intricate chemical mechanism underlying the viability, and 
ultimately, the senescence, of living animal cells. 

So far as the nutrition of spermatozoa is concerned, semen 
resembles more a suspension of microorganisms in a nutrient 
medium, than other animal tissues which rely for their nutrients 


xiv The Biochemistry of Semen 

on the blood supply. Nature has endowed the spermatozoa with the 
means of very efficient utilization of extraneous sources of energy, 
such as are accessible to the sperm cells either in their natural 
environment, the seminal plasma, or in the artificial storage media. 

As will be evident from what follows later, the present century 
has witnessed much that is new in the field of semen biochemistry. 
By and large, however, the situation is not very different from what 
it was two centuries ago, when Charles Bonnet addressed the follow- 
ing remarks about spermatozoa to Spallanzani: 

'They are, of all animalculi of liquids, those which have most 
excited my curiosity: the element in which they live, the place of 
their abode, their figure, motion, their secret properties; all, in a 
word, should interest us in so singular a kind of minute animated 
beings. How are they found there, how are they propagated, how 
are they developed, how are they fed, and what is their motion? 
What becomes of them when the liquid they inhabit is reabsorbed 
by the vessels and returned to the blood? Why do they appear only 
at the age of puberty; where did they exist before this period? Do 
they serve no purpose but to people the fluid where they are so 
largely scattered? How far are we from being able to answer any of 
these questions! And how probable it is, that future age will be as 
ignorant of the whole, as our own!' 


77?^ Two Components of Semen: 
Spermatozoa and Seminal Plasma 

Spermatozoa. Spermatogenesis and sperm 'ripening'. Sperm transport in 
the female reproductive tract and 'capacitation'. Structural and chemical 
characteristics of the sperm-head, middle-piece and tail. 

Seminal plasma. Secretory function of male accessory glands. Prostatic 
secretion. Seminal vesicle secretion. Physiological significance of seminal 
plasma. Prostaglandin, vesiglandin, and certain other pharmacodynamic- 
ally active substances. Coagulation and liquefaction. 

'Whole semen' as ejaculated, generally appears as a viscous, creamy, 
slightly yellowish or greyish fluid, and consists of spermatozoa or 
'sperm', suspended in the fluid medium, called seminal plasma; its 
composition depends in the first place, on the proportion of sperm 
and plasma, and is further determined by the size, storage capacity, 
and secretory output of several different organs which comprise the 
male reproductive tract. The volume of the ejaculate and the con- 
centration of spermatozoa or the 'sperm density' in ejaculated semen, 
vary widely from one species to another, as seen from Table 1. A 
single ram ejaculate for instance, amounts to 0-7-2 ml. only, but is 
distinguished by a very high sperm density, 2-5 million per f.i\. 
semen; when subjected to high-speed centrifugation, ram semen 
separates, on the average, into about two-thirds of seminal plasma 
and one-third of firmly packed sperm. Boar semen ejaculates on the 
other hand, may reach a volume of as much as 500 ml; this is not 
due to spermatozoa, but to the seminal plasma generated in very 
capacious accessory organs (Plate III); a sperm density not exceed- 
ing 100,000 cells //^l. is quite usual for boars, and even lower sperm 
densities would still be regarded as normal. In man, the average 
volume of a single ejaculate is about 3 ml., but the sperm density 
is frequently less than 100,000 cells///!., so that only a small portion 
of the ejaculate, much less than 10%, is represented by the sperm 
and the rest is seminal plasma. 


The Biochemistry of Semen 

Table 1 . Species differences in volume and sperm density of 
ejaculated semen 

Volume of single ejaculate 

Sperm density in semen 





Most common 
value 1 

Normal variations 

Average value 



50 ' 

200 000- 600 000 

400 000 



5 000 000-8 000 000 

6 000 000 




25 000- 300 000 

100 000 




300 000-2 000 000 

1 000 000 


0-2-1 -5 


50 000-6 000 000 

3 500 000 




1000 000-9 000 000 

3 000 000 




30 000- 250 000 

70 000 




50 000- 150 000 

100 000 




100 000-2 000 000 

700 000 




2 000 000-5 000 000 

3 000 000 




30 000- 800 000 

120 000 




7 000 000 

The two components of semen, sperm and seminal plasma, differ 
in their origin, composition and function, and must be considered 
separately, in much the same sense as for instance, blood corpuscles 
and blood plasma. 

Early investigators of semen were, not unnaturally, fascinated by 
the spermatozoa, and, with the aid of such optical instruments as 
were available to them, concentrated their efforts upon the elucida- 
tion of the structural details of spermatozoa. But it is very much to 
their credit that they have not entirely neglected the seminal plasma. 
Thus, the letter in which Antoni van Leeuwenhoek reported in 1 677 
to the Royal Society on sperm motion, also contains the earliest 
description of spermin crystals in the seminal plasma. Louis Nicolas 
Vauquelin, the author of the first treatise on the chemical composi- 
tion of semen (Experiences sur le sperme humain, 1791), fully appre- 
ciated the separate existence of sperm and seminal plasma; the same 
was true of his followers, among them Friedrich Miescher, whose 
collected writings, published in 1897, contained much new informa- 
tion concerning not only spermatozoa but the seminal plasma as 

The work of Miescher and his contemporaries, however, dealt 
largely with fish spermatozoa, and even during the early decades of 

77?^ Two Components of Semen 3 

the present century, research on semen was, on the whole, confined 
to fish and generally to animals in which fertilization takes place 
externally, and which provide the experimental material in con- 
veniently large quantities. The tardy progress of research on the 
spermatozoa and seminal plasma of birds and mammals was due 
in the main to the difficulty of securing enough material for experi- 
mental purposes; however, more rapid advances were made soon 
after Elie Ivanov (1907) and several other pioneers in the field of 
artificial insemination, perfected the technique of semen collection 
from domestic animals. The widening practice of artificial insemina- 
tion for breeding purposes on a large scale, early revealed the need 
for improved standards of sperm evaluation and in this way pro- 
vided a powerful stimulus for morphological as well as chemical 
investigations on semen. At the same time, clinical enquiries into the 
causative and diagnostic aspects of human infertility also pointed 
to serious gaps and deficiencies in the knowledge of the physiology 
of human semen. 

The last two decades have witnessed rapid advances in the applica- 
tion of laboratory methods of semen analysis to the study of the 
manifold causes underlying male sterility and subfertility, and there 
is a steadily increasing number of publications on this subject, which 
has been comprehensively reviewed on several occasions. Some of 
these articles and monographs refer specifically to man (Joel, 1942; 
Hammen, 1944; Hotchkiss, 1945; Hinglais and Hinglais, 1947; 
Farris, 1950; Lane-Roberts, Sharman, Walker, Wiesner and Barton, 
1948; Bayle and Gouygou, 1953; Longo, 1953; Williams, 1953), while 
others deal with various animals (Gunn, 1936; Burrows and Quinn, 
1939; Anderson, 1945; Bonadonna, 1945; Perry, 1945; Walton, 1945; 
Milovanov and Sokolovskaya, 1947; Van Drimmelen, 1951; Millar 
and Ras, 1952). In addition, much valuable information on sperm 
physiology in general, indispensable alike to those engaged in human 
and in animal research, will be found in the writings of Marshall 
(1922), Hartman (1939), Chang and Pincus (1951) and Walton (1954), 
as well as in the published records of various symposia and con- 
ferences held under the auspices of such bodies as the Biochemical 
Society {Biochemistry of Fertilization and the Gametes, 1951), the 
New York Academy of Sciences {Biology of the Testes, 1952), the 
Ciba Foundation {Mammalian Germ Cells, 1953), the National 

4 The Biochemistry of Semen 

Committee on Maternal Health {Diagnosis in Sterility, 1946; The 
Problem of Fertility, 1946; Studies on Testis and Ovary, Eggs and 
Sperm, 1952), the American Society for the Study of Sterility 
(official journal: Fertility and Sterility) and the British Society for the 
Study of Fertility {Proceedings). 


Spermatogenesis and sperm 'ripening'' 

Spermatozoa (Plate I and Fig. 1) originate in the testis from the 
germ or spermatogenic cells of the seminiferous epithelium in the 

Galea capitis 






Nucleus (poscerior part) 


Axial filament 

Fig, 1. Diagrammatic representation of a spermatozoon. 

course of spermatogenesis, a process of stepwise proliferation and 
transformation, distinguished by the successive stages of sperma- 
togonia, primary spermatocytes, secondary spermatocytes, and 
spermatids. The present knowledge concerning the chemical changes 

The Two Components of Semen 5 

which take place during spermatogenesis is defective and rests almost 
entirely on histochemical observations. 

In several species so far investigated, spermatogonia and sperma- 
tocytes have been shown to have a cytoplasm which is basophilic, 
in distinction to mature spermatozoa which exhibit only a faint 
coloration of the flagellum. Cytochemical studies carried out by 
Brachet (1944, 1947) have shown that the affinity of the sperma- 
togonia and spermatocytes for basic dyes is due to ribonucleic acid, 
and cytochemical as well as spectrophotometric studies (Caspersson, 
1939) point to the fact that spermatogenesis involves a progressive 
disappearance of ribonucleic acid from the developing sperm cell. 
In ejaculated spermatozoa of the bull, Vendrely and Vendrely (1948) 
using the analytical methods of Schmidt and Thannhauser (1945) 
and Schneider (1945), found a content of 0-2x1 0~^ mg. ribonucleic 
acid per sperm cell, that is fifteen times less than the corresponding 
value for deoxyribonucleic acid. An analysis of mature ram sperma- 
tozoa carried out in our laboratory with the Markham-Smith 
chromatographic procedure (1949) which is based on the identifica- 
tion of uridylic acid in an acid hydrolysate of ribonucleic acid, 
failed to reveal the presence of uridylic acid. As to the origin of 
ribonucleic acid in the spermatogonia and spermatocytes, a study of 
the spermatogenesis in Asellus aquaticus (Vitagliano and de Nicola, 
1948) suggests that ribonucleic acid is not elaborated in the develop- 
ing gametes themselves but is secreted by the surrounding cells and 
then absorbed and utilized by the germ cells. 

Two other processes associated with spermatocytic development 
are: the progressive decline of alkaline and acid phosphatase activity 
(assessed histochemically) in the nuclei (Krugelis, 1942; Wolf, Kabat 
and Newman, 1943), and a simultaneous disappearance of glycogen. 
Both the Sertoli cells and the spermatogonia abound in glyco- 
gen, which also occurs, although in a smaller concentration, in the 
primary spermatocytes (Montagna and Hamilton, 1951; Elftman, 
1952; Long and Engle, 1952; Mancini, Nolazco and Baize, 1952). 
But the secondary spermatocytes and the spermatids give practically 
no cytochemical reactions for glycogen, and in the mature sperma- 
tozoa the glycogen content is exceedingly low: in ejaculated ram 
semen 'glycogen' content, i.e. the alkali-resistant polysaccharide 
which behaves like glycogen on ethanol-precipitation and which 

6 The Biochemistry of Semen 

yields on hydrolysis glucose (as determined by glucose oxidase) 
seldom exceeds 01%, and may be as little as 0019% (Mann, \9A6b). 
Similarly, in sea-urchin sperm {Echinus esculentus), the ethanol- 
precipitable, glycogen-like material separated from sperm and 
analysed after acid hydrolysis by means of glucose oxidase, repre- 
sents no more than 004% on a wet-weight basis (Rothschild and 
Mann, 1950). Even oysters, in which as much as one-third of the dry 
body weight may consist of glycogen, produce spermatozoa which 
when ripe, contain no more than 1 % glycogen on a dry-weight basis 
(Humphrey, 1950). 

Yet another phenomenon accompanying spermatogenesis is a 
significant change in the distribution of lipids. In the deer (Wislocki, 
1949) and in the rat (Lynch and Scott, 1951), sudanophilic material 
is concentrated chiefly in the Sertoli cells, but in man (Montagna, 
1952) a high content of lipids is characteristic alike of the Sertoli 
cells as well as the cytoplasm of spermatogonia and of primary 
spermatocytes. Similarly, in certain invertebrates, as for example, 
Lithobius forficatiis (Monne, 1942), lipids form a highly characteristic 
component of the cytoplasm in spermatocytes. These cytoplasmic 
lipids are usually birefringent and give positive reactions for steroids. 
In ejaculated spermatozoa, at any rate those of mammals, the lipids 
are confined largely to certain definite regions such as the 'mito- 
chondrial sheath' of the middle-piece and the so-called lipid capsule; 
these will be described in more detail later. 

The changes initiated by spermatogenesis continue during the stay 
of spermatozoa in the epididymis, and form a part of the 'ripening' 
process. The metabolism of epididymal spermatozoa which are often 
immotile, but capable of long survival, is as yet only poorly under- 
stood. Guinea-pigs and rabbits, for example, can remain fertile 
for some weeks after the ligation of the ductuli efferentes, and in bats 
spermatozoa have been detected in the cauda epididymis as late as 
seven months after the cessation of spermatogenesis. 

A striking change associated with the process of sperm ripening 
in the epididymis is the migration of a drop-like swelling of sperm 
cytoplasm called the 'kinoplasmic droplet' and believed to contain 
some lipid material; when one examines spermatozoa from the caput 
epididymidis of a mouse for example, the kinoplasmic droplet is 
usually situated close to the proximal (anterior) end of the middle- 

The Two Components of Semen 1 

piece, but by the time the spermatozoa have reached the cauda 
epididymidis and are nearing the vas deferens, the droplets take up 
a position at the distal (posterior) end of the middle-piece (Merton, 
1939; Fig. 2). Finally, they tend to disappear altogether and are 
seldom found in ejaculated sperm, except m certain abnormal cases 
(Plate I). Some authors regard the kinoplasmic droplet as no more 
than a remnant of spermatid cytoplasm devoid of special signi- 
ficance, but there are those who believe that it plays an important 
role by nourishing the spermatozoon during the passage through the 
epididymis, before the sperm cells establish contact with an extra- 
cellular source of nutrient material, in the form of seminal plasma. 
The disappearance of the kinoplasmic droplet is but the final 
stage in the process of gradual shrinkage and 'dehydration' of proto- 
plasm which accompanies both spermatogenesis and ripening, and 
from which the 'ripe' spermatozoon ultimately emerges as a cell 
with a highly condensed nucleus and very little cytoplasm. Associated 
with the diminution of protoplasm is a progressive loss of water 
and a corresponding increase in the specific gravity of the sperm cell. 
Lindahl and Kihlstrom (1952) suspended equal numbers of bull 
spermatozoa in a series of aqueous solutions of 
the methylglucamine salt of 'umbradil' (2 : 5-di- 
iodine-4-pyridone-A^-acetic acid), the lightest of 
which (sp. g. 10918, osmotic pressure 18 atm.) 
had a lower specific gravity than any of the 
spermatozoa, the heaviest (sp. g. 1-3519, osmotic 
pressure 220 atm.) being of about the same 
specific gravity as the 'densest' spermatozoa; 
these sperm suspensions were centrifuged in 
haematocrit tubes so that all spermatozoa with 
a specific gravity exceeding that of the medium, 
formed a sediment in the graded capillary part of 
haematocrit tubes. The specific gravity of bull 

Fig. 2. ''Ripening'' process in the epididymis of the 
mouse; (a) spermatozoon from the caput epi- 
didymis with proximal kinoplasmic droplet; 
(b) spermatozoon from the cauda epididymis 
with distal kinoplasmic droplet. 

(Merton, 1939) 


8 The Biochemistry of Semen 

spermatozoa determined in this manner ranged from 1-240 to 1-334; 
there was a negative correlation between the mean specific gravity 
and the percentage of unripe spermatozoa, that is those which 
still possessed the kinoplasmic droplet; in each experiment, the 
concentration of unripe spermatozoa was significantly higher in 
the 'floating', than in the sedimenting, fraction, the specific gravity 
of the unripe sperm cells being less than that of the ripe ones. 

To some extent, the specific gravity of spermatozoa may be 
accounted for by the high concentration of deoxyribonucleoprotein 
in the sperm nucleus, but in a large measure it is also due to the state 
of 'dehydration' which is characteristic of the sperm protoplasm 
and its protein constituents. Hand in hand with the high specific 
gravity goes a remarkably high refractive index and light-reflection 
power of the spermatozoa. In general, the refractive index of most 
living animal cells lies between 1-350 and 1-367, corresponding to a 
10-20% concentration of solids; but in human spermatozoa exam- 
ined by the immersion method, Barer, Ross and Tkaczyk (1953) 
obtained values corresponding to a content of almost 50% solids. 
Nephelometric measurements of light reflection carried out with 
bull semen samples containing a varying percentage of 'unripe' 
spermatozoa, showed that the capacity of the sperm cell to reflect 
light increases with ripening (Lindahl, Kihlstrom and Strom, 1952); 
there appears to be a close relationship between the light-reflecting 
power of sperm and the characteristic 'luminosity' of the surface 
of spermatozoa under dark-field illumination, which, in all prob- 
ability, is due to the 'waterlessness' of the lipid capsule surrounding 
the ripe sperm cell. 

Sperm transport in the female reproductive tract and ' capacitation' 

There is evidence that the process of sperm ripening is not halted 
at ejaculation but proceeds in the female reproductive tract, where 
the sperm cell undergoes a definite change, called capacitation, 
before it becomes capable of penetrating the egg surface (Austin, 
1951; Chang, 1951; Austin and Braden, 1952; Thibault, 1952). It is 
quite likely that the success which some early investigators had in 
achieving fertilization with artificially inseminated epididymal sper- 
matozoa, was due to the continuation of sperm ripening processes 
in the female reproductive tract. 

The Two Components of Semen 9 

Whether the semen is ejaculated into the uterus (sow), or into the 
cervix or vagina (cow, rabbit), a certain time is always required for 
the passage of spermatozoa to the oviducts and for their accumula- 
tion in adequate numbers at the site of fertilization. The time needed 
for some of the spermatozoa at any rate, to arrive at their goal may 
be relatively short; a quarter of an hour or less, in the rat (Blandau 
and Money, 1944), cow (VanDemark and Moeller, 1951) and ewe 
(Starke, 1949; Dauzier and Wintenberger, 1952); a matter of a few 
minutes in the rabbit (Lutwak-Mann, unpublished). This indicates 
that in these animals the spermatozoa are conveyed to their final 
destination thanks to certain concomitant movements of the 
female tract and do not depend exclusively upon their own motility. 
However, from the moment of arrival in the ovarian tube, time 
must elapse before the sperm cell is capable of fertilizing the egg. 
In the rabbit, ovulation takes place about ten hours after copulation, 
and presumably, spermatozoa require this period of time to undergo 
complete 'capacitation'. As Chang (1951) has shown, rabbit sperma- 
tozoa placed in the Fallopian tubes soon after ovulation, penetrate 
a larger proportion of eggs if they had been previously kept for about 
five hours in the uterus of another doe. According to Austin (1951), 
rat spermatozoa injected into the periovarian sac of the rat after 
ovulation, do not begin to enter the eggs until some five hours later. 
The processes of sperm maturation and capacitation are linked in 
some as yet not fully understood manner, with the survival of sperm 
in the female tract. In higher mammals this period is usually limited 
to one or two days, but the 'longevity' of bird sperm is remarkable, 
and in bats and the terrestrial isopode Armadillidium vulgare the 
spermatozoa are said to survive in the female tract for many months, 
in certain insects even for years. In insects, however, this striking 
behaviour of spermatozoa is probably bound up closely with certain 
other peculiarities of sperm transport: in many instances, the sperma- 
tozoa are conveyed to the female not in a free fluid medium, but are 
enclosed in a sac or 'spermatophore' which is deposited in the 'bursa 
copulatrix' or in the vagina; from there, after the sac has been 
emptied, they move on to the 'spermatheca', a pouch which serves 
as a special storage organ for the spermatozoa, where they remain 
till the time of fertilization. 

It must also be remembered that not all spermatozoa present in 

10 The Biochemistry of Semen 

a given ejaculate survive for the same length of time. In higher 
mammals for instance, of the many hundreds of millions of sperm 
cells, only a minute fraction, not more than a few thousand, reach 
the site of fertilization, and ultimately only a single spermatozoon is 
responsible for the fertilization of the ovum. 

Structural and chemical characteristics of the sperm-head, middle- 
piece, and tail 

In the majority of species, including man, mature spermatozoa 
have a filiform structure owing to the presence of a flagellate append- 
age, although non-flagellar forms of sperm cells are not uncommon 
in certain lower animals, for example, among Crustacea and nema- 
todes. This peculiar filiform structure determines to a considerable 
extent, the remarkable permeability of the sperm cell, which is per- 
haps best illustrated by the so-called 'leakage' phenomenon, that is, 
the remarkable ease with which even large molecules such as cyto- 
chrome c or hyaluronidase can detach themselves from the sperm 
structure and pass into the extracellular environment. The high 
degree of permeability explains the speed with which exchange re- 
actions can take place between the spermatozoa and the surrounding 
medium, whether this be the seminal plasma or an artificial pabulum; 
moreover it makes it possible for certain intermediary enzymic 
reactions such as those involved in the phosphorylative breakdown 
of carbohydrate, to be demonstrated directly in intact spermatozoa, 
without cell disintegration which is an unavoidable prerequisite in 
studies on the intermediary enzymes of other animal tissues. This 
does not necessarily apply to all enzymes and the failure to demon- 
strate an enzyme in intact sperm cells must not be taken as evidence 
of its absence, particularly so in the case of mammalian sperma- 
tozoa which are resistant to the action of most plasmolysing agents, 
including water. 

The principal morphological features of spermatozoa have been 
established largely in the last century with the help of the ordinary 
light microscope, by pioneers such as Ballowitz, Jensen, Meves, 
Retzius and others, but many more details have emerged since as a 
result of the application of new techniques, particularly those of 
histochemistry (Marza, 1930; Popa and Marza, 1931; Brachet, 1944; 
Leblond, Clermont and Cimon, 1950; Leuchtenberger and 




a. Normal bull semen; photographed in ultraviolet light at 2750 A. 

Mag. X 2700. 
h. Semen from an infertile bull; the spermatozoa are 'unripe' and show 

kinoplasmic droplets at the anterior ends of the middle-pieces; 

nigrosin-eosin stain. 


c. Rat spermatozoon. 

d. Rat spermatozoon with a kinoplasmic droplet at the posterior end of 

the middle-piece. 

e. Rat spermatozoon. Mag. x 1500. 

(By courtesy of Dr. C. R. Austin and Dr. J. L. Hancock) 

The Two Components of Semen 11 

Schrader, 1950; Wislocki, 1950; Friedlaender and Fraser, 1952; 
Hancock, 1952; Melampy, Cavazos and Porter, 1952) and electron 
microscopy (Seymour and Benmosche, 1941; Harvey and Anderson, 
1943; Schmitt, 1944; Bretschneider and Iterson, 1947; Bretschneider, 
1949^, b\ Grigg and Hodge, 1949; Hodge, 1949; Randall and Fried- 
laender, 1950; Bayle and Bessis, 1951; Friedlaender, 1952; Challice, 
1953; Bradfield, 1954). 

In a typical flagellar spermatozoon (Plate I and Fig. 1) it is usually 
possible to distinguish three regions, viz. sperm-head, middle-piece 
and tail, but even among closely related species, one encounters an 
extraordinary diversity of form, size and structure. Moreover, on 
examining the semen from a single individual, one often finds in 
addition to the normally shaped spermatozoa, a variety of 'degener- 
ate', 'abnormal' or 'immature' forms which represent every con- 
ceivable deviation from the normal cell structure, from 'tapering' 
and 'double' cells with a double head or tail, to 'giant' and 'monster' 
cells containing several nuclei and several tails in a mass of cyto- 
plasm. Although a high degree of sperm abnormality is undoubtedly 
associated with subfertility, normal semen is seldom completely 
uniform, and human semen for example, is reckoned to contain 
as a rule, at least 20% of abnormal forms (Pollak and Joel, 1939; 
Harvey and Jackson, 1945; Hotchkiss, 1945; Lane-Roberts et al., 
1948; Williams, 1950). In the bull (Williams and Savage, 1927; 
Lagerlof, 1934; Bishop, Campbell, Hancock and Walton, 1954) 
and stallion (Bielanski, 1951), the percentage of abnormal forms in 
semen is similarly high, in the ram on the other hand, it appears to 
be much less. 

The shape of the head in a normal spermatozoon varies greatly; 
it is ovoid in the bull, ram, boar, and rabbit, it resembles an elong- 
ated cylinder in fowl and has the form of a hook in the mouse and 
rat; in the human species, the sperm-head appears as a flattened, oval 
body, about 4-6 ^ long, 2-6 /< wide, and 1-5 f-i thick, which is com- 
pressed at the anterior pole into a thin edge. 

The main part of the head is occupied by the nucleus, filled by 
closely-packed chromatin which consists largely of deoxyribonucleo- 
protein and gives a positive Feulgen (nucleal) reaction with Schiff"'s 
fuchsin-sulphurous acid reagent. The anterior part of the nucleus 
is covered by a cap-like structure known as the acrosome. The 

12 The Biochemistry of Semen 

latter gives no positive Feulgen reaction but stains with the Schiff 
reagent after exposure to the oxidizing action of periodic acid as 
demonstrated in the sperm of the hemipteran insect, Arvelius albo- 
punctatus (Leuchtenberger and Schrader, 1950) and in bull sperm 
(Hancock, 1952). According to McManus (1946) and Hotchkiss 
(1948), the 'periodic acid Schiff reaction' (PAS) is due to the pre- 
sence of carbohydrates, and the chemical groups which react with 
fuchsin-sulphurous acid are the aldehydes formed from 1 : 2 glycol 
groupings by oxidation with periodic acid: 

I I 

R— C C— R + HIO4 -> 2R— CHO 

The acrosomal material is not glycogen as it does not react with 
iodine and is not affected by treatment with amylase. It cannot be 
hyaluronic acid because it resists the action of hyaluronidase. The 
possibility that it may be related to hyaluronidase itself still remains 
to be investigated. There has also been a tendency to regard it as a 
mucopolysaccharide, without however, sufficient evidence. Special 
precautions are called for in the preparation of spermatozoa for the 
PAS reaction. Structural changes in sperm cells, such as occur for 
example, after rapid cooling ('temperature shock'), may render the 
acrosomal material unresponsive to the periodic acid-Schiff reagent. 
It is not improbable that the acrosomal 'polysaccharide' is either 
decomposed or detached from the head of a mature spermatozoon; 
this is borne out by some microscopic observations on changes which 
take place in the acrosome during the period of senescence and death 
of the sperm cell. Several investigators have described in sperma- 
tozoa yet another cap, a loose protoplasmic structure, named 'galea 
capitis' (also 'acrosome cap', 'Kopfkappe' or 'capuchon cephalique') 
which envelops the apical part of the sperm-head and can break 
away spontaneously to form a so-called 'spermatic veil' or 'floating 
cap' (Williams and Savage, 1925; Blom, 1945). However, whereas 
most authors including Williams (1950) regard the acrosome proper 
and the galea capitis as two distinct structural entities, some con- 
sider them to be identical, and Hancock (1952) for instance, is con- 
vinced that there is only one acrosomal structure and, that the de- 
tachable cap arises through post-mortem changes, and is the result 
of swelling and loosening of the acrosome itself. The separation of 

The Two Components of Semen 1 3 

the galea capitis can be conveniently followed by the India-ink 
staining technique of Blom (Fig. 3) whose studies indicate that the 
phenomenon occurs most frequently in degenerating spermatozoa, 
for example, in ejaculates obtained after long periods of abstinence. 
When stained by the Gomori technique, the galea capitis or at least 
the region near the tip of the head, shows a positive reaction for acid 
phosphatase. On the whole, however, phosphatase activity, 'acid' 
and 'alkaline' alike, is much more intense in the seminal plasma 
than in the spermatozoa. Moreover, as it is rather difficult to 

Fig. 3. Schematic representation of the head of a bull spermatozoon before 
and after detachment of the galea capitis {India-ink method). 

a, pars posterior, b, pars intermedia, c, pars anterior with the galea capitis 

in situ, q, the 'bare' pars anterior after detachment of the galea, Cg, galea 

capitis detached from the head. 

(Blom, 1945) 

remove from the spermatozoa, even by exhaustive washing, all 
adhering traces of seminal plasma, the possibility of contamination 
with plasma phosphatase must be taken into account when con- 
sidering the occurrence of phosphatase in the spermatozoa themselves. 
The narrow region which connects the sperm-head with the 
middle-piece is known as the neck (or neck-piece), which is the most 
vulnerable and fragile part of the spermatozoon. The entire neck 
region is bounded, however, by a membrane which continues over 
the head and middle-piece. In the neck, close to the base of the 
sperm nucleus, is situated the centrosome which marks the beginning 
of the 'axial filament', the central core of both the middle-piece and 
tail. The axial filament consists of a number of fine long fibrils which 

14 The Biochemistry of Semen 

run uninterruptedly through the whole length of the middle-piece 
and tail. These fibrils probably represent the main contractile ele- 
ment of the sperm cell, responsible for the whip-like lashing of the 
tail. In most species investigated so far by means of the electron 
microscope, eleven fibrils have been identified; two of these, which 
occupy the central position, are sensitive to the action of water and 
digestive proteolytic enzymes, whereas the remaining nine fibrils 
which form an 'outer cylinder' around the 'central pair', are remark- 
ably resistant to the action of plasmolysing and digestive agents, 
and even prolonged proteolysis with pepsin or trypsin fails to disrupt 
them; these fibrils also resist effectively attempts at solubilization by 
means of salt solutions, acids and weak bases. 

The finer structure of the individual fibrils is still a matter of active 
investigation. In the case of mammalian spermatozoa, doubling of 
fibrils has been observed, at any rate in the middle-piece, and in 
addition to the outer cylinder of nine fibrils, another, so-called 
inner cylinder has been described, consisting of nine, much thinner 
fibrils. The precise chemical nature of the fibrillar protein is un- 
known. A certain resemblance to muscular contraction prompted 
Engelhardt (1946) to ascribe to the contractile substance of sperma- 
tozoa myosin-like properties, and to sperm adenosine-triphos- 
phatase the role of 'spermosin'. However, this c^aim remains at 
present unsubstantiated since it was not accompanied by satisfac- 
tory evidence that the spermatozoa used for the experiments, were 
really free from phosphatases, especially the powerful adenosine- 
triphosphatase, of seminal plasma. 

In the middle-piece (or midpiece) which in the human sperma- 
tozoon is about the length of the sperm-head though only one-tenth 
as wide, the axial filament is surrounded by the 'broad helix', also 
called 'spiral body' or 'mitochondrial sheath'. This lipid-rich struc- 
ture, which is believed to be derived from mitochondria, has the 
shape of a broad paired thread, wound helicoidally round the 
'outer cylinder' of sperm fibrils. It is here that the cytochrome- 
cytochrome oxidase system of spermatozoa is believed to be con- 
centrated. The junction between the middle-piece and tail is marked 
by the presence of a ring centriole. 

The tail or 'flagellum' in the human spermatozoon is about ten 
times the length of the middle-piece and lacks the 'broad helix' 



Broken end of tail from a bull spermatozoon, showing the tuft of fibrils 
of the axial filament, and the helical structure of the tail sheath; 
I 1 indicates 1 /x. 

(By courtesy of Prof. L. H. Bretschneider and Dr. Woutera van Iterson) 

The Two Components of Semen 15 

but has instead the much thinner 'tail sheath' or 'cortical helix* 
which terminates a short distance before the end of the tail, exposing 
the terminal portion of the axial filament, that is the end-piece. In 
mammalian spermatozoa, the tail sheath appears as a helicoidally 
wound cord; when the tail of the spermatozoon is broken, one can 
see, protruding from the cortical sheath, the brush-like fibrils of the 
axial filament, and at this point it is also possible to distinguish the 
helical structure of the tail sheath (Plate II). In fowl spermatozoa on 
the other hand, there is no evidence of a 'cortical helix', and the axial 
filament is encased in an amorphous sheath which is easily dis- 
rupted by distilled water, causing the axial filament to fray into 
fibrils. In addition to the various fibrous cortical systems, the sperm 
cell of many species, including man and the higher mammals, is 
protected externally by a lipid layer or capsule ('manteau lipidique') 
evident especially around the tail, and composed of a layer of 


Seminal plasma, the extracellular fluid which provides the medium 
and vehicle for spermatozoa, originates in the accessory organs of 
reproduction and varies in composition according to species. In 
lower animals it may be so scarce that the emitted semen takes the 
form of a very thick lump of spermatozoa, closely packed together. 
There is little seminal plasma in bird semen and even among some 
of the mammals, but on the whole, the higher mammals, including 
man, produce a relatively dilute semen with a considerable propor- 
tion of seminal plasma. 

Secretory function of male accessory glands 

The seminal plasma is a composite mixture of fluids secreted by 
organs which in the higher species comprise the epididymides, the 
seminal ducts or vasa deferentia, ampullae, prostate, seminal vesicles 
(or seminal glands), Cowper's glands and certain other glands 
located in the wall of the urethral canal. Until a little while ago, the 
secretory function of the male accessory organs remained obscure 
chiefly owing to lack of information about the chemical nature of 
the various secretions. More recently, however, several substances 


The Biochemistry of Semen 

have been discovered and identified in the accessory secretions, such 
as citric acid by Schersten in 1929, prostatic phosphatase by Kutcher 
and Wolbergs in 1935, fructose by Mann in 1945, phosphoryl- 
choline by Lundquist in 1946, ergothioneine by Leone and Mann, 
and inositol by Mann in 1951, and glycerylphosphorylcholine by 

Rabbit Rat 

Fig. 4. Diagrammatic outline of male accessory organs to illustrate the 
localization of fructose {shaded areas). 

Am, ampullae. SV, seminal vesicle. Pr, prostate. VP, ventral prostate. 

DLP, dorsolateral prostate. Pp, glandulae paraprostaticae. GV, glandula 

vesicularis. CG, coagulating gland. 

Diament, Kahane and Levy in 1952 (for details concerning the 
secretory function of male accessory organs see: Mann and Lutwak- 
Mann, 1951ft; Lutwak-Mann, 1951). 

Owing to the complex nature of the seminal plasma the physio- 
logist or biochemist is forced to adopt a distinct approach when 
investigating any one of the accessory gland secretions. There are 
several instances where male accessory organs which, though pre- 
viously believed on the basis of similar embryonic origin or related 



A, prostate; B, seminal vesicle; C, vas deferens; D, Cowper's gland; E, 
caput epididymidis; F, cauda epididymidis; G, testis; H, bladder. 
Scale in inches. 

The Two Components of Semen 17 

morphological structure, to be anatomically and even functionally 
'homologous', were later shown to differ greatly in their chemical 
secretory activity. This is particularly true of the secretions of the 
prostate and the seminal vesicle, two organs which in the majority 
of higher species provide the bulk of the seminal plasma; their 
localization within the reproductive tract of several species is illus- 
trated in Plate III and Fig. 4. 

The prostatic secretion 

This differs in many ways from other secretions of the mammalian 
body, and its composition shows considerable species variations. 
Much study has been devoted to the human and canine prostatic 
fluids; both are colourless and usually slightly acid, about pH 6-5 
(Huggins, 1947; Zagami, 1940) and both are remarkable for the 
almost complete absence of reducing sugar. They abound, however, 
in several strong proteolytic enzymes; the human prostatic fluid 
contains a fibrinolysin so powerful, that 2 ml. of prostatic fluid can 
liquefy 100 ml. clotted human blood in 18 hr. at 37°; dog prostatic 
fluid is distinguished by its ability to destroy fibrinogen, but it is 
relatively inactive towards clotted blood (Huggins and Neal, 1942). 
The prostate secretes a diastase (Karassik, 1927), and a /3-glucuroni- 
dase which is more active in man than in dog (Talalay, Fishman and 
Huggins, 1946; Huggins, 1947). 

The prostatic secretion represents the main source of citric acid 
and of acid phosphatase for whole human semen; and the analysis 
of these two constituents provides a most convenient 'chemical 
indicator test' for the assessment of the functional state of the 
human prostate. There is much more citric acid and acid phospha- 
tase in the human, than in the canine, secretion; thus, the citric acid 
content is less than 30 mg./lOO ml. in dog, as against 480-2680 
mg./lOO ml. in the human fluid; acid phosphatase activity in dog 
corresponds to about 28 King-Armstrong units/100 ml. in the 
'resting' or spontaneously voided prostatic secretion, and 104 
units/100 ml. in the 'stimulated' secretion obtained by parasym- 
pathetic stimulation, whereas the prostatic secretion of a normal 
adult man may contain up to 3950 units/1 ml. (Gutman and 
Gutman, 1941; Huggins, 1947). 

The concentration of osmotically-active substances in the 

1 8 The Biochemistry of Semen 

prostatic fluids of man and dog is shown in Fig. 5. In the human 
secretion (Huggins, Scott and Heinen, 1942), the average values for 
cations, expressed in m-equiv./l. water, are: sodium 156, potassium 
30, and calcium 30; for anions: citrate 156, chloride 38, bicarbonate 
8, and phosphate 1. In a pilocarpine-stimulated dog prostatic fluid 
(Huggins, Masina, Eichelberger and Wharton, 1939) the base con- 
























Fig. 5. Diagram of osmotically active substances in prostatic fluid. 

(Huggins, 1947) 

sists of sodium 162, and potassium 5; the anions chloride 156, and 
bicarbonate 1-7 m-equiv./l. In man, the prostatic secretion also 
provides the main source of calcium; the so-called Niederland re- 
action, which depends on the formation of characteristic needle- 
shaped crystals in human semen heated with dilute sulphuric acid, 
is probably due to calcium sulphate (Niederland, 1931, 1935; 
Ziemke, 1931). 
^ Among the chemical peculiarities of the prostate gland is its 

The Two Components of Semen 19 

rather high content of zinc. The first to observe this, and to comment 
on the possible role of zinc in reproduction, was Gabriel Bertrand; 
in the two analyses of human prostate carried out by Bertrand and 
Vladesco (1921) there was found 9-4 and 11-3 mg. Zn per 100 g. 
fresh tissue, or 49 1 and 53 1 mg. Zn per 100 g. dry weight. More 
recently, Mawson and Fischer (1951, 1952, 1953) in Canada, found 
that the mean zinc content of the human prostate gland was 68-2 mg. 
Zn/100 g. dry wt., which is in considerable excess of zinc content 
in human liver, muscle, brain, testis or blood. These investigators 
state that the zinc present in the human seminal plasma is derived 
chiefly from the prostatic secretion. 

A considerable number of studies have been carried out with the 
rat prostate (Fig. 4). In the rat, there is a distinct anatomical and 
functional difference between the so-called ventral prostate which 
secretes only citric acid, and the dorso-lateral prostate which pro- 
duces both citric acid and fructose (Humphrey and Mann, 1948, 
1949). In the dorso-lateral prostate itself, however, it is possible 
to distinguish three smaller regions, the dorsal or median portion 
which does not contribute citric acid, and two lateral lobes which 
are rich in citric acid (Price, Mann and Lutwak-Mann, 1949). It is 
the dorso-lateral prostate, and more specifically, its two lateral lobes 
which contain much more zinc than any other soft tissue of the rat, 
and which at the same time exhibit carbonic anhydrase activity 
almost equal to that of blood (Mawson and Fischer, 1952); whereas 
however, the carbonic anhydrase in the rat lateral prostate accounts 
for no more than one-tenth of the total zinc content, in blood 
erythrocytes this enzyme is well known to correspond closely to 
the bulk of the zinc content (KeiUn and Mann, 1940). 

The protein content of the prostatic secretion is low, less than 1 % 
in man, and a certain proportion of the protein-like material present 
in the secretory fluid is composed of 'proteoses' which are not preci- 
pitated by trichloroacetic acid. Another feature of the prostatic 
secretion is its elevated content of certain free amino acids, the 
presence of which is probably the outcome of a combined action 
of proteolytic and transaminating enzymes in the glandular tissue 
(Barron and Huggins, 1946a; Awapara, \952a, b). Human prostatic 
adenoma contains in 100 g. tissue 50 to 200 mg. glutamic acid in 
addition to several other amino acids. The average content of amino 

20 The Biochemistry of Semen 

acids in protein-free filtrates of ground prostatic adenoma or dog 
prostate, expressed in terms of millimoles/100 g. tissue, is 34 and 
39, respectively. The ventral lobe of the rat prostate contains in a 
free state nearly all known amino acids, and in addition phosphoryl- 
ethanolamine, taurine, glutathione, and glutamine. The dorso-lateral 
lobe on the other hand, in contrast to the ventral prostate, has a 
much lower content of most amino acids and lacks completely iso- 
leucine and threonine; it may be added here that it also responds 
differently to castration and to hormones. 

The seminal vesicle secretion (Tables 2 and 3) 

In several species, including the rat, guinea-pig and bull, the 
seminal vesicles alone contribute more fluid than the rest of the 

Table 2. Some characteristic constituents of the seminal vesicle 
secretion in man, bull, boar, rat and guinea-pig 

(In each species, two individuals were examined, and results expressed 
as averages (mg./lOO ml.); the human vesicular secretion may have been 
contaminated with prostatic fluid. Ascorbic acid was determined as dinitro- 
phenylhydrazone, and the value represents the sum of the oxidized and 
reduced form.) 






Dry weight 






Ascorbic acid 






Citric acid 












Inorganic phosphorus 






Acid-soluble phosphorus 












* In rat, fructose is secreted by the coagulating gland, an organ adjacent 
to the seminal vesicle (see Fig. 4). 

accessory glands together. Whereas however, in the rat and guinea- 
pig the seminal vesicles conform to the pattern of true thin-walled 
and large 'vesicles', the bull seminal vesicles are more correctly des- 
cribed as seminal 'glands', with multiple lobes of glandular tissue 
which surrounds a system of ramified secretory ducts. The size and 
storage capacity of the seminal vesicles, and their secretory output, 

The Two Components of Semen 21 

are subject to individual variations, which are particularly conspicu- 
ous in man. But the storage capacity of the human vesicles is small 
indeed in comparison with that of the bull or boar. In certain mam- 
mals such as the dog or cat, the seminal vesicles are altogether 
absent. In the rabbit, a combined anatomical and biochemical study 
of the reproductive system has shown that the two organs known as 
glandula seminalis and glandula vesicularis, develop from the same 
diverticulum of the Wolffian duct, and possess a common urethral 
outlet, so that both these glands together may be regarded as homo- 
logous to the seminal vesicles proper of other mammals (Davies and 
Mann, 1947«, b; Mann, 1947). 

Table 3. Composition of the boar seminal vesicle secretion 
(m^./lOO ml.) 

(Analysis of 670 ml. seminal vesicle fluid representing the pooled 
secretions collected from three boars; sp. g. 1046; pH 7-2.) 

Dry weight 



soluble in 66% ethanol 

18 635 

6 565 

12 070 

5 347 









Inorganic phosphorus 
Inorganic sulphur 
Total nitrogen 
Non-protein nitrogen 



1 548 







Total anthrone-reactive 

Total aminosugar 



3 053 

Total sulphur 




Lactic acid 


Citric acid 


Total phosphorus 
Acid-soluble phosphorus 


22 The Biochemistry of Semen 

Compared with the prostatic fluid, the seminal vesicle secretion 
is usually less acid, sometimes distinctly alkaline, has a higher dry 
weight and contains more potassium, bicarbonate, acid-soluble 
phosphate and protein; the latter is to a large extent precipitable by 
trichloroacetic acid but there is also some 'proteose' as shown for 
example, by the study of the seminal vesicle proteins in the goby 
Gillichthys mirabiUs (Young and Fox, 1937). But the most remark- 
able feature of the seminal vesicle secretion is its unusually high 
content of reducing substances. 

The normal seminal vesicle secretion is usually slightly yellowish 
but occasionally, especially in man and bull, it can be deeply pig- 
mented. The yellow pigmentation is probably of composite origin 
but much of it is due to flavins which cause the vesicular secretion and 
seminal plasma to fluoresce strongly in ultraviolet light. Brochart 
(1952) observed that when strongly yellow coloured samples of bull 
seminal plasma are exposed to sunHght, the colour tends to dis- 
appear within a short time and lumiflavin is formed. Leone (1953) 
has shown that at least part of the flavin content of the bull seminal 
vesicle secretion is due to adenine-isoalloxazine dinucleotide, 
associated with xanthine oxidase. The highest content of total flavin 
which I was able to record in the bull seminal vesicle secretion, was 
750 /^g./lOO ml.; in eight samples of bull seminal plasma there was 
from 47 yf^g./lOO ml. (in an almost colourless specimen) to 480 
^g./lOO ml. flavin (in a particularly deeply pigmented specimen). 
There can be little doubt that the flavin associated with the strongly 
yellow-coloured specimens of bull semen is due principally to the 
seminal plasma, and not to the spermatozoa. The sperm cells them- 
selves, however, contain also some flavin. In washed bull sperma- 
tozoa, there is some 30 [ig. riboflavin/g. dry weight (Lardy and 
Phillips, 1941c), and in whole bull semen, particularly in the less 
coloured samples, a substantial portion of flavin may be derived 
from the spermatozoa (VanDemark and Salisbury, 1944). 

In addition to the yellow pigment, the seminal plasma sometimes 
contains a brownish haematin pigment; this occurs in cases of 
'chronic haemospermia', a condition occasionally met with in man 
and attributed to haemorrhagic changes in the seminal vesicles 
(McDonald, 1946). 

Potassium in a high concentration occurs in the vesicular secretion 

The Two Components of Semen 23 

of several species, including man (20 mM), bull (100 mM) and 
boar (300 mM). In the latter, the ionic equilibrium on the cationic 
side is set up chiefly by potassium, with citric acid as the main anion; 
the concentration of sodium is much less than that of potassium; 
chlorides are conspicuous by their almost total absence, a phe- 
nomenon infrequently encountered in other normal body fluids 
(Table 3). Another unusual feature of the boar vesicular secretion is 
its high content of inositol which varies from 2 to 3% and accounts 
for something like one-third of the total dialysable material; inositol 
together with citrate, contributes substantially to the osmotic pres- 
sure of the vesicular secretion (Mann, 1954). 

The reducing power of the vesicular secretion is one of its most 
characteristic chemical properties. Two kinds of reducing sub- 
stances are present. One group is made up of substances which 
are capable of reducing silver nitrate, iodine and 2 : 6-dichlorophenol 
indophenol in the cold. They are always present in the protein-free 
filtrate from the vesicular secretion and seminal plasma but 
their chemical nature varies from one species to another. At 
one time, the reducing property was generally attributed to 
ascorbic acid, in the secretions of the guinea-pig (Zimmet and 
Sauser-Hall, 1936; Zimmet, 1939), bull (Phillips, Lardy, Heiser and 
Ruppel, 1940; Jacquet, Cassou, Plessis and Briere, 1950) and man 
(Nespor, 1939; Berg, Huggins and Hodges, 1941). According to 
Phillips and his co-workers, bulls of high fertility produce semen 
containing 3 to 8 mg. ascorbic acid per 100 ml., whereas low-fertility 
bulls may have less than 2 mg./lOO ml.; these workers have also 
claimed that in certain bulls it was possible to enhance the fertility 
by parenteral administration of ascorbic acid. More recent studies, 
however, based upon chemical methods of purification and identi- 
fication, have proved that ascorbic acid rarely accounts for the total 
reducing power of semen towards dichlorophenol indophenol. In 
the boar especially, ascorbic acid has been shown by Mann and 
Leone (1953) to account but for a small fraction of seminal reducing 
power and the bulk of the reducing material was found to consist 
or ergothioneine, a substance which owes its reducing power to a 
sulphydryl group (Leone and Mann, 1951; Mann and Leone, 1953). 
The properties and functions of ergothioneine will be discussed 
later (p. 174). 

24 The Biochemistry of Semen 

The presence of the other kind of reducing substances can be 
detected in protein-free filtrates from semen and seminal vesicle 
fluid, by heating with sugar reagents, such as for example, cupric 
hydroxide. In this category belongs fructose, the physiological sugar 
of semen. 

Huggins and Johnson (1933) were the first to observe that the 
reducing sugar of human semen is derived from the secretion of the 
seminal vesicles but is absent in the prostate. Similar findings were 
made with the bull (Bernstein, 1937), boar (McKenzie, Miller and 
Bauguess, 1938) and ram (Moore and Mayer, 1941). The identifi- 
cation of the seminal sugar as fructose (Mann, 1946a, b, c) opened 
the way for detailed studies of the fructose-generating capacity of 
the accessory tissues (Fig. 4). It was shown that in several species 
fructose is secreted either by the seminal vesicles or by functionally 
related organs (Mann, 1946c; 1947; l94Sa, b). This made it possible 
to use the chemical assay of fructose in semen as an indicator of the 
relative contribution made by the seminal vesicles towards the make- 
up of the whole semen. It must be pointed out, however, that a 
certain small amount of fructose is also produced by the ampullar 
glands and in some species, by certain other accessory organs (see 
p. 138). 

The physiological function of the seminal plasma 

From time to time doubt is expressed as to whether the individual 
accessory gland secretions or even the entire seminal plasma, have 
any essential role to fulfil in the process of reproduction; the more 
so, since in some anmials such as the guinea-pig or rabbit, it is 
possible to induce pregnancy by the artificial insemination of epi- 
didymal spermatozoa. It is however, arguable as to how much sig- 
nificance may be ascribed to such experiments, and it is certain that 
the natural mating process could scarcely be expected to function 
smoothly and efficiently without the provision of seminal plasma 
as a normal diluent and vehicle for the thick mass of closely packed 
epididymal spermatozoa; no more could the blood corpuscles act 
as oxygen carriers in vivo, without the blood plasma. 

Furthermore, the seminal plasma exerts a distinct stimulating 
effect on sperm motility. In part, this is due simply to the 'dilution 
effect', a phenomenon which is described fully elsewhere (see p. 73). 

The Two Components of Semen 25 

To a considerable extent, however, the activation by seminal plasma 
has been shown to be linked with the occurrence of specific sub- 
stances in the different accessory gland secretions. In certain insects, 
Bombyx mori for example, the spermatozoa are believed to acquire 
their full fertilizing capacity only after activation by the secretion 
of the lower portion of the male tract, the so-called glandula pros- 
tatica (Wigglesworth, 1950). Many investigators have studied the 
activating influence of the prostatic secretion on mammalian sper- 
matozoa (Steinach, 1894; Hirokawa, 1909; Ivanov, 1929; Sergijewski 
and Bachromejew, 1930), and in some instances, e.g. the dog, have 
claimed it to be species-specific (Ivanov and Kassavina, 1946), By 
no means all of these experiments, however, have been carried out 
under satisfactorily controlled conditions and, as Huggins (1945) 
thinks, in many cases it is impossible to exclude the action of non- 
specific factors such as acceleration of sperm motility by certain ions 
or by changes in the gas tension. 

Much discrimination is equally needed in appraising certain 
results obtained with the epididymal secretion. Here, however, the 
problem involves not so much the 'activation', as the 'ripening' and 
'life-prolonging', effects on the spermatozoa (Tournade and Mer- 
land, 1913; Stigler, 1918; Braus and Redenz, 1924; Hammond and 
Asdell, 1926; Young, 1929, 1930; Young and Simeone, 1930; Lanz, 
1931, 1936; Gunn, 1936; Gunn, Sanders and Granger, 1942). Most 
investigators agree that in some special way the epididymis is 
adapted for the long-term storage of spermatozoa; but whereas some 
like Braus and Redenz (1924), attribute to the epididymal secretion 
a specific role in the 'ripening' process, others deny it such a func- 
tion. In the seminal vesicle fluid, we seem to be confronted with an 
activating effect on spermatozoa different from that exerted by the 
prostatic and epididymal secretions, one which is specifically bound 
up with the presence therein of fructose which provides a source of 
nutrient material for the sperm cells. 

Another facet of the physiological function of the seminal plasma 
concerns certain characteristic pharmacological effects of the acces- 
sory gland secretions and the role of seminal plasma in the remark- 
able process of semen coagulation and liquefaction. 

26 The Biochemistry of Semen 

Prostaglandin y vesigiandin, and certain other pharmacodynamically 
active substances 

Among the more striking pharmacological effects of seminal 
plasma are a depressor action on blood vessels, and a stimulation 
of isolated smooth-muscle organs such as the uterus and the intes- 
tines. Both these effects which have been studied in great detail by 
von Euler (1934«, b\ 1935, 1937, 1939, 1949) and Goldblatt (1933, 
1935^?) are due in all probability not to a single substance but to the 
combined action of several constituents of seminal plasma, including 
choline and two substances which Euler has named 'prostaglandin* 
and 'vesigiandin'. So far, only prostaglandin has been purified. 
The purest preparation obtained from ram prostate glands has been 
found by Bergstrom (1949) to be nitrogen-free, and to contain an 
unsaturated acidic substance which absorbs strongly ultraviolet light 
with a maximum at 280 m//. When assayed on the isolated intestine 
of rabbit, 1/^g. of this substance exhibited the same activity as the 
crude extract from 500 mg. prostatic tissue. Assuming that the sub- 
stance is pure, the total content of prostaglandin in 100 kg. prostate 
glands must be of the order of 25-50 mg. To prepare this quantity, 
one would require the glands from 20,000 rams. 

The physiological significance of prostaglandin and vesigiandin in 
reproduction processes is unknown but it has been suggested that 
they represent some sort of 'automatic regulator' which controls 
the voiding of the secretions from the prostate and the seminal 
vesicle, respectively. The idea of chemical stimulation by secretory 
products is based among others, on observations that the emptying 
of the prostate and seminal vesicle leads to a decreased ability of 
these glands to contract which persists until enough of freshly 
formed secretion has accumulated. 

A pharmacodynamic influence of the seminal plasma upon some 
parts of the female reproductive tract has also been envisaged but 
the evidence is derived mainly from experiments on isolated organs. 
It remains questionable whether any of the effects exerted by the 
seminal plasma on the uterus and oviduct in vitro, also occur in the 
female reproductive tract in vivo (Barnes, 1939; Asplund, 1947). 

Kurzrok and Lieb (1931) found on adding 1 ml. of human seminal 
fluid to a strip of human uterus suspended in a 100 ml. bath, either 

The Two Components of Semen 27 

an increase or a decrease in spontaneous contractions. Cockrill, 
Miller and Kurzrok (1935) observed that those specimens of human 
semen which were capable of enhancing uterine contraction, caused 
an inhibition after having been exposed for half an hour to pH 10; 
at pH 11 all specimens became inactive. Moreover, the effect was 
potentiated by eserine and suppressed by atropine. The observed 
action corresponded to that of about 100 ^g. acetylcholine per ml. 
semen. About that time, the occurrence of a powerful oxytocic 
substance in the human seminal plasma was demonstrated by Gold- 
blatt {\92>5b) who also found that the activity was destroyed by 
short boiling of the seminal plasma with either OlN-NaOH or 
OlN-HCl. When assayed on the guinea-pig's uterus, 1 ml. of human 
seminal plasma showed approximately the same oxytocic activity 
as 0-4-0-6 mg. of histamine. Euler (1937) believes that the oxytocic 
activity is due to prostaglandin which in his experiments stimulated 
strips of human uterus and also isolated uterus as well as uterine 
strips from several species including the cow, rabbit, guinea-pig and 
rat. Asplund (1947) determined the total content of 'contractive sub- 
stance' in 155 specimens of human semen which he assayed on the 
rabbit intestine in vitro, with purified prostaglandin as the standard. 
He came to the conclusion that the effect of semen on the isolated 
intestine must be attributed to a combined action of prostaglandin, 
choline, and at least one other substance which produces a very 
rapid increase in tonus and is unaffected by atropine. There was no 
correlation between the total content of 'contractive substance' in 
seminal plasma and the motility and viability of spermatozoa. 

In addition to the remarkable vaso-dilation and contraction of 
plain muscle, the seminal plasma and the accessory gland secretions 
exhibit a characteristic pressor activity towards blood vessels. In 
1906 Jappelli and Scafa found that parenterally administered extracts 
of the canine prostate produced an increased blood pressure in the 
dog and stimulated the respiration. A similar effect was observed by 
Thaon (1907) after the intravenous injection of prostatic extracts into 
rabbits but in his experiments the rise in blood pressure was usually 
followed by a fall. There are indications that the pressor action of 
the prostatic extracts is due to adrenaline-like substances demon- 
strated in semen and accessory glands by CoUip (1929), v. Euler 
(1934Z>), Bacq and Fischer (1947), and Brochart (1948fl) (see p. 181). 

28 The Biochemistry of Semen 

Coagulation and liquefaction 

Semen is ejaculated in a liquid or semi-liquid form. In some animal 
species such as the bull and the dog, it remains liquid, but in others 
it tends to gelate or coagulate shortly after ejaculation. Human 
semen clots immediately after ejaculation only to liquefy again a 
little later; until that happens the spermatozoa do not become fully 
motile. For this reason the examination of human spermatozoa 
should be postponed until at least twenty minutes after the emission 
of semen (McLeod, 1946<2; Sunmons, 1946). Quite fresh boar semen 
usually contains only small lumps of gelatinous material somewhat 
resembling tapioca; on standing, however, the lumps increase and 
merge into a semi-solid gelatinous mass which may take up half or 
more of the entire ejaculate. Gelation of semen can also be observed 
in the stallion. Even more striking is the clotting phenomenon in 
the rodents. The major part of a rabbit ejaculate collected by means 
of an artificial vagina often consists of a colourless transparent gel. 
In rats and guinea-pigs, semen coagulation leads to the formation 
after mating, of the so-called bouchon vaginal or vaginal plug, which 
probably prevents the back-flow of semen from the vagina and 
assists the passage of spermatozoa through the cervix into the uterus. 
In a study of sperm transport in the rat, Blandau (1945) has shown 
that the ejaculate fails to pass through the uterine cervix if the 
coagulation of semen is aboUshed by ligation of the seminal vesicle 
and coagulating gland ducts. The copulatory plug has also been 
described in Insectivora (mole and hedgehog), Chiroptera {Rhinolo- 
phidae and Vespertilionidae) and in Marsupialia (Camus and Gley, 
1899; Courrier, 1927; Eadie, 1939, 1948a, b; Engle, 1926; Rollinat 
and Troussart, 1897; Stockard and Papanicolaou, 1919). In most 
animals the vaginal plug is due to the clotting of the semen itself but 
in some, namely in the opossum (Hartman, 1924) and in the bat 
Vesperuga noctula (Courrier, 1925; Grosser, 1903), its occurrence 
involves the coagulation of female secretions by the seminal plasma. 
In the honey bee, the escape of semen from the female reproductive 
tract is prevented by the so-called mucus plug; this is formed by the 
material ejected from the mucus glands of the drone towards the end 
of ejaculation (Laidlaw, 1944). 

In most species the substrate for gel formation consists of protein- 

The Two Components of Semen 29 

like material secreted by the seminal vesicles (Bergmann and Leuc- 
kart, 1855; Bischoff, 1852; Hensen, 1876; Landwehr, 1880; Lataste, 
1888; Pittard, 1852; Stockard and Papanicolaou, 1919), the enzyme 
however, responsible for the coagulation is absent in the vesicles 
themselves and comes into contact with the protein substrate only 
in the course of ejaculation. The first to recognize the enzymic 
nature of the coagulating agent were Camus and Gley (1896, 
1899, 1907, 1921, 1922) who named it 'vesiculase'. It was shown by 
Walker (1910) that the source of the coagulating enzyme in small 
rodents is the so-called 'coagulating gland', which lies adjacent to 
the seminal vesicle. The clotting power of the secretion from the 
coagulating gland is such that 1 part is sufficient to clot 20,000 parts 
of the seminal vesicle secretion. This quantitative relationship can 
be demonstrated in vitro by collecting the two fluids separately and 
then mixing them after suitable dilution (Moore and Gallagher, 

The liquefaction of human semen which follows its coagulation, is 
also an enzymic process. Fibrinolysin and fibrinogenase, two pro- 
teolytic enzymes of semen (see p. 1 14), are believed to play a part in 
this process. 


Chemical and Physical Properties 
of Whole Ejaculated Semen 

Species and individual variations in the composition of semen. Pre- 
sperm, sperm-containing, and post-sperm fractions in the ejaculate. 
Criteria for the rating of semen quality. Optical and electrical properties 
of semen. Viscosity, specific gravity, osmotic pressure, and ionic equi- 
librium. Hydrogen ion concentration and buffering capacity. Metabohsm 
of semen and its relation to sperm density and motility; glycolysis; 
methylene-blue reduction test; respiration. 

Species and individual variations in the composition of semen 
A CHARACTERISTIC feature of whole semen is the variability of its 
composition not only among different species but also between in- 
dividuals of the same species (Table 4). Somewhat less pronounced 
but still significant are the variations in the concentration of some 
of the semen constituents in the same individual (Table 5). All this 
is not altogether unexpected since both the spermatogenic activity 
of the testes and the secretory function of the male accessory organs 
are subject to considerable physiological fluctuations of hormonal 
origin and are influenced by factors such as light, temperature, 
season, state of nutrition etc. The variability of semen composition 
is the reason why repeated examination of whole semen, even if 
restricted to a single experimental subject, and carried out under 
identical experimental conditions, need not yield the same quanti- 
tative results, as might, for example, an analogous series of blood 
analyses in the same individual. Therefore, an accurate estimate of 
semen quality in any one individual cannot be formed on the basis 
of a single analysis and involves several examinations. 

In Table 5 are shown individual fructose variations in the semen 
of man, bull, ram and rabbit. The analytical results are given in 
terms of concentration (mg./lOO ml. semen) and of absolute content 


Chemical and Physical Properties of Semen 3 1 

(mg, /ejaculate). This method of expressing the findings of semen 
analysis is very useful particularly in the evaluation of pathological 
abnormalities in the composition of semen. The data in Table 5, 
especially those relating to rabbit semen, show that the variations 
in the absolute content of fructose per single ejaculate are much 
less than those in concentration; the reason being that a rabbit 


- 3-0 



/I \ 


/ \ 



- 2-5 


/ i ' Vi 




_ 8 


- 2-0 



/ \ '/ 


/ \ * 

/ \* 


1 6 

- 1-5 






- 1-0^ 


1 1^ 
, / 



- 0-5 

1 1 1 1 


1-5 % 

012 34S6 789 10 

Time (weeks) 

12 13 14v 

Fig. 6. Relation between volume (( 

and content of fructose (0 — 
and citric acid {% •)/« rabbit semen 

(Mann & Parsons, 1950) 

•) of ejaculate 


ejaculate is usually made up of two main portions, gel and fluid, of 
which the latter is relatively constant whereas the quantity of gel 
is subject to considerable fluctuation. Fructose, however, is con- 
fined chiefly to the fluid portion and consequently, its absolute 
content in semen varies little. On the other hand, citric acid, another 

Table 4. Species differences in the chemical composition of semen 

(Results are average values (range in brackets) expressed in mg./lOO ml. 
except for COg content (ml. /1 00 ml.); for information on the composition 
of dog and cock semen see appendices (6) and (7).) 






Dry weight 







Chloride (CI) 


















































Total nitrogen 



















Uric acid 





















Lactic acid 






Citric acid 










Total phosphorus 1 1 2 











Lipid phosphorus 6 











(1) Human semen. Data on dry weight, total nitrogen, and electrolytes 
(excluding inorganic phosphate): Huggins, Scott and Heinen (1942); COg: 
Sheldovsky et al. (1940); urea: Goldblatt (1935^); Lactic acid: Lundquist 
(1949Z>). Remaining data are our own; the results on non-protein nitrogen, 
fructose, and citric acid content are based on analysis of semen from ten 
individuals. The ash content of human semen is about 0-9% (Slovtzov, 
1902, 1916); the ash contains 3% sulphur (Albu, 1908). According to 
Infantellina (1945) human semen contains 30 mg./lOO ml. glutathione. 
The values reported for ascorbic acid are 12-8 mg./lOO ml. (Berg, Huggins 
and Hodges, 1941) and 2-6-3-4 mg./lOO ml. (Nespor, 1939); using Roe's 
method, we found 10-2 and 12-4 mg./lOO ml. in two specimens. 

(2) Bull semen. Data on electrolytes (except magnesium and inorganic 
phosphate): Bernstein (1933); COg: Shergin (1935); remaining data are our 
own, based on analysis of ejaculates from ten bulls (average volume of 
ejaculate 4-5 ml.; sperm concentration 985,000/jul.). Other constituents of 
bull semen include (mg./lOO ml.): thiamine 0-028-0-152, riboflavin 0-152- 
0-306, pantothenic acid 0-230-0-466, and niacin 0-248-0-554 (VanDemark 
and Sahsbury, 1944). Ehlers et al. (1953) reported the following mean 
values based on the analysis of 663 samples of semen (mg./lOO ml.): 
fructose 552 (S.D. 169), citric acid 724 (S.D. 192). For additional data see 
Rothschild and Barnes (1954). 

(3) Ram semen. With the exception of the CO2 content (Shergin, 1935), 
the data are our own, based on analysis of material pooled from ten 
ejaculates (average volume of single ejaculate 1-2 ml.; 2,940,000 sperm/jitl.). 

(4) Boar semen. Data on dry weight, electrolytes and total nitrogen: 
McKenzie, MUler and Bauguess (1938); remaining data our own. Addi- 
tional information in Table 7. 

(5) Stallion semen. Data on dry weight, electrolytes, total phosphorus 
and CO2 content from Slovtzov (1916), Bernstein (1933), Shergin (1935) 
and Milovanov (1938); remaining data our own. 

(6) Dog semen. Much work has been done on the dog prostatic secretion 
(see text). Whole semen has been analysed by Slovtzov (1916) and the 
following composition found (mg./lOO ml.): dry weight 2450, albumin, 
globulin and nucleoprotein 866, mucoprotein 57, and lipid 182. Electro- 
lytes have been examined by Bernstein (1933); dog semen is distinguished 
by a high content of chloride (620-657 mg./lOO ml.). Fructose and citric 
acid occur only in traces. 

(7) Cock semen. In material from six pooled ejaculates, 5,100,000 
sperm//il., we found (mg./lOO ml.): 57 total anthrone-reactive carbo- 
hydrate, of which 4 was fructose and 41 glucose (determined by glucose 
oxidase); 44 total phosphorus, of which 27 was acid soluble; 2 ammonia. 
In six individual specimens of cock semen we found from 7-7 to 81 
mg./lOO ml. glucose but never more than 4 mg./lOO ml. fructose. 



The Biochemistry of Semen 

Table 5. Individual variations in the level of fructose in semen 
(Ejaculates collected at weekly intervals.) 








100 ml.) 


100 ml.) 


100 ml.) 


100 ml.) 
















































































































characteristic chemical component of rabbit semen, is found largely 
in the gel, and its varying content reflects the variability of the 
latter (Fig. 6). 

Frequency of ejaculation also affects the composition of individual 
samples of semen. Table 6 shows the results of a so-called exhaustion 
effect on the semen of a bull (Mann, 1948«); in this experiment, eight 
ejaculates were collected from the same animal within 63 min., at 
7-10 min. intervals. As a result of the multiple collections, the sperm 
density fell from 1,664,000 cells///l. in the first, to 98,000 cells/^ul. 
in the last, ejaculate. This decrease, however, was not accompanied 
by a corresponding diminution of fructose concentration, which was 
much the same in the first (760 mg./lOO ml.) and last (690 mg./lOO 
ml.) ejaculate. But it must be remembered that the seminal vesicles 
of a bull differ from those of man and other mammals by their 
exceptional capacity: in some bulls it is possible to recover up to 
50 ml. of secretory fluid from the seminal vesicles, enough to provide 
at least a dozen fructose-rich ejaculates. 

Chemical and Physical Properties of Semen 35 

Table 6. Ejfect of frequency of collection on sperm density and on 

concentration of fructose and lactic acid in fresh bull semen 

(Mann, 1948a) 


Time of 



Volume of 



Sperm density 

(thousands/ 1/d. 



(mg./lOO ml. 


Lactic acid 

(mg./lOO ml. 

















































Pre-sperm, sperm-containing, and post-sperm fractions in the ejaculate 
To assess correctly the composition of individual specimens of 
semen it is essential to bear in mind the fact that in some species 
(e.g. man, boar, stallion) the different portions of semen follow 
one another in a definite order of sequence. This has been demon- 
strated by the so-called split-ejaculate method which depends on the 
collection and analysis of separate fractions of the same ejaculate 
according to the time of delivery from the urethra. In man, the 
ejaculation is initiated by the secretion of Cowper's glands, the pros- 
tatic secretion is delivered next, to be followed by the sperm and the 
vesicular secretion (Broesike, 1912; Huggins and Johnson, 1933; 
MacLeod and Hotchkiss, 1942; Lundquist, 19496; Pryde, 1950); 
according to Lundquist, in man the prostatic secretion contributes 
from 13 to 32% and the vesicular secretion from 46 to 80% of the 
whole ejaculate. 

In the boar, an animal with a protracted period of ejaculation, 
the semen consists of two portions, gel and liquid. McKenzie et al. 
(1938) calculated that 15-20% of the liquid portion is derived from 
the seminal vesicles, 2-5% from the epididymis, 10-25% from 
Cowper's gland, and the rest is made up by the urethral glands 
secretion. We have made a similar investigation (Mann and Glover, 
1954) using estimations of sperm concentration in ejaculated and 
epididymal semen for the assessment of the epididymal contribution, 
and the chemical determinations of fructose, citric acid and ergo- 
thioneine in the ejaculated semen and in the vesicular secretion as 

36 The Biochemistry of Semen 

a means for the evaluation of the contribution of the seminal vesicles. 
From the results of this study (Table 7) it can be seen that in 
ejaculated semen (liquid portion), the sperm concentration was 3% 

Table 7. Composition of ejaculated semen, epididymal semen, and 
seminal vesicle secretion of the same boar 

(Four ejaculates were collected from the same boar, at weekly intervals. 
The boar was then killed, and the epididymal semen and vesicular secretion 
collected. For chemical analysis, the fluid portions were used, separated 
from sperm and gel by centrifugation. In the case of ejaculated semen, the 
means (and standard deviation) are given per single ejaculate.) 





from the 





Total volume (ml.) 

375 (±24) 



Sperm concentration 

(thousand//iil.) in 

the liquid portion 

108 (±9) 


Volume of fluid portion (ml.) 

292 (±12) 



Composition of the fluid portion 

(mg./lOO ml.) 


9 (±0-5) 




17 (±M) 



Citric acid 

173 (±9) 



Lactic acid 

21 (±3) 



Total dry weight 




Dry wt. of non-dialysable 





Total phosphorus 




Acid-soluble phosphorus 
















Hexosamine (after acid 




(t^) of that present in the 

epididymis, and the concentrations of 

fructose, ergothioneine and citric acid, were 

20-9% (tV), 20-9% (il), 

and 20-8% (|^), respectively, of those 

present in 

the seminal 

vesicle secretion. In semen collected from the boar by the 'split- 
ejaculate method' it is possible to distinguish clearly three distinct 
fractions. According to McKenzie et al. (1938), the pre-sperm 

Chemical and Physical Properties of Semen 37 

fraction comprises 5-20%, the sperm-rich fraction 30-50%, and the 
post-sperm fraction 40-60% of the total ejaculate. The pre-sperm 
fraction which is ejaculated first, consists of a watery, often dis- 
coloured, more or less sperm-free secretion, probably of urethral 


' i '■ 


Af / 

V '• 



N '• 


l\ 1 ^ ^\ 

W \ i^B 


// 1 ^' 




1 L ' 


V \ ' 


W\ ,. 

L , V* , \a 1 

12 3 4 5 6Min. 

Fig. 7. Composition of boar semen fractions collected by the 'split-ejaculate 
method* at half-a-minute intervals. 

A, sperm number xlO^/fraction (• #); B, fructose content, 

mg./fraction (O O); C, ergothioneine content, m./fraction 

(• •). 

(Glover & Mann, 1954) 

38 The Biochemistry of Semen 

origin. Within a few minutes, that is followed by a sperm-containing 
fraction which may also contain some gel-like material. As a rule 
however, the gel forms a distinct fraction and is voided shortly after 
the sperm fraction. Moreover, these three fractions, which complete 
an ejaculation 'wave', may be succeeded by a second 'wave', also 
fractionated. The two waves together may last up to 30 min., and 
represent in fact, two successive ejaculates. By the estimation of 
sperm concentration coupled with the chemical determination of 
fructose, citric acid, and ergothioneine in the various fractions, it is 
possible to show that during the ejaculation the seminal vesicle 
secretion follows immediately upon the delivery of the spermatozoa, 
and is found mainly in the sperm-containing fraction (Glover and 
Mann, 1954; Fig. 7). Occasionally, however, the ejaculation of 
semen is incomplete and the semen does not include the later 
fractions. When this happens, for example in the stallion, certain 
normal constituents such as fructose or citric acid, may be missing 
altogether. Obviously, such incomplete ejaculations create a further 
complication in the assessment of analytical results obtained with 
semen, at any rate in those species in which the ejaculation is a 
fractionated one. But even in the bull where under physiological 
conditions ejaculation appears to be instantaneous, the 'split- 
ejaculate method' applied by Lutwak-Mann and Rowson (1953) 
with the aid of electric stimulation, demonstrated the occurrence in 
electrically-induced ejaculates, of at least two distinct fractions; of 
these, the first was a copious sperm-free fraction, slightly viscous, 
colourless, and of urethral origin, in which there was no fructose or 
citric acid and very little protein; the next was a creamy sperm- 
containing fraction, followed by, or more often mixed with, a yel- 
lowish-coloured post-sperm fraction which represented an almost 
pure secretion of the seminal vesicles, with a characteristically high 
content of fructose, citric acid, and 5-nucleotidase. 

Criteria for the rating of semen quality 

General medical and veterinary experience indicates that the suc- 
cessful fertihzation of the ovum and initiation of pregnancy, while 
it is brought about primarily by the spermatozoa present in an 
ejaculate, demands nevertheless the attainment of certain qualita- 
tive and quantitative standards by the semen. We should therefore, 

Chemical and Physical Properties of Semen 39 

be able to define what constitutes normal fertile semen, and what 
criteria if any, can be applied in the appraisal of ejaculated semen. 
It may be stated at once that in spite of the wealth of information 
gained by past and present students of semen, there is as yet no single 
seminal characteristic known, which alone could serve as the means 
of judging 'male fertility'. The best criterion of the fertilizing capacity 
of spermatozoa is of course, the actual ability to fertilize the ovum. 
This however, cannot be regarded as a laboratory test, until the 
in vitro fertilization of the ovum has actually been accomplished 
and the quantitative aspects of the process developed. 

In the practice of artificial insemination of cattle, male fertility 
continues to be assessed on the basis of the 'conception rate'. At 
the artificial insemination centres, inseminated cows which have 
not been 'returned' by the farmers for re-insemination within three 
months or so, are presumed to have conceived, and the proportion 
of presumed pregnancies, expressed as the percentage of the total 
of the first inseminations, is referred to as 'conception rate'. Nearly 
one-third of the cow population of England is now bred by artificial 
insemination, and among these the conception rate averages at least 
60%. There is considerable evidence, however, that a substantial 
proportion of cows 'returned' for re-insemination, may have also 
conceived but that pregnancy terminated at an early stage through 
faulty ovum implantation or embryonic death. 

Apart from the test based upon accomplished fertilization, the 
means available at present for the evaluation of semen quality 
include the histological and the physico-chemical methods. 

Histological examination of semen involves procedures such as 
the determination of sperm concentration or 'density' (number of 
spermatozoa per \fj\. or 1 ml. of semen) with a cytometer (Walton, 
1927; Weisman, 1942); differential count of abnormal forms of 
spermatozoa (Lagerlof, 1934; Harvey and Jackson, 1945; Lane 
Roberts et al., 1948; Williams, 1950); bacteriological examination 
(Gunsalus, Salisbury and Willett, 1941; Kelly, 1947; Foote and 
Salisbury, 1948; Almquist, Prince and Reid, 1949; Wu, Elliker and 
McKenzie, 1952-3); determination of the incidence of dead sperma- 
tozoa by means of 'live-dead staining' methods (Lasley, Easley and 
McKenzie, 1942; Lasley and Bogart, 1943; Madden, Herman and 
Berousek, 1947; Crooke and Mandl, 1949; Blom, 1950; Mayer, 

40 The Biochemistry of Semen 

Squiers, Bogart and Oloufa, 1951; Ortavant, Dupont, Pauthe and 
Roussel, 1952; Campbell, Hancock and Rothschild, 1953); micro- 
scopic assessment of the degree of motility, either directly in semen 
(Harvey and Jackson, 1945; McLeod, \9A6a; Emmens, 1947; Farris, 
1950) or by the 'cervical mucus penetration test', in which a drop of 
semen is placed on the microscope slide next to cervical mucus and 
the passage of spermatozoa through the mucus is followed by micro- 
scopic observation (Barton and Wiesner, 1946; Harvey and Jackson, 
1948). The determination of the concentration of motile spermatozoa 
in a semen sample is generally held to be the criterion most clearly 
correlated with the actual fertility rate; but even this relationship 
occasionally fails to give a true picture, and motile spermatozoa 
are by no means always fertile. 

Physico-chemical methods of semen analysis depend on the deter- 
mination of a wide range of physical, chemical and metabolic 
characteristics of semen, related to the physiological function of the 
sperm and the seminal plasma. Here belong methods for the 
measurement of certain optical properties of semen such as the 
light-scattering and light-absorption power, electrical conductivity 
and impedance changes; specific gravity, osmotic pressure; hydrogen 
ion concentration, buffering capacity; occurrence of semen-specific 
metabolites such as fructose, citric acid, ergothioneine, and inositol, 
and various enzymes such as hyaluronidase and certain phospha- 
tases; and finally, the rate of anaerobic and aerobic metabolic 
processes in semen expressed in terms of fructolysis index, respira- 
tion rate or methylene-blue reduction time. 

Optical and electrical properties of semen 

An optical property closely related to sperm concentration is the 
turbidity of semen. In the past, determinations of sperm concen- 
tration were based on microscopic sperm-counts but more recently 
these have been partly replaced by turbidimetric measurements, 
which can be carried out quickly with suitably diluted samples either 
in a visual comparator by direct comparison with opacity standards, 
or in a photoelectric absorptiometer, to provide values for the rela- 
tive light transmission of semen (Burbank, 1935; Comstock and 
Green, 1939; Henle and Zittle, 1942; Salisbury et al. 1943; Roth- 
schild, 19506). The turbidimetric methods, however, it must be 

Chemical and Physical Properties of Semen 41 

remembered, rest on the assumption that although light is absorbed 
both by sperm and seminal plasma, the scattering of light is due 
exclusively to the former, so that only light-scattering as such, is 
related directly to sperm density. In practice, the light-scattering and 
light-absorption due to the spermatozoa predominates so much 
over the light-absorption of the seminal plasma that under properly 
controlled conditions the error due to the presence of the latter can 
be neglected. This applies certainly to the semen of sea-urchin, bull 
and ram, but need not necessarily be true of other species, in which 
the seminal plasma itself shows a considerable degree of opaqueness. 
Furthermore, the spermatozoa present in a given ejaculate may 
differ in their light-reflecting capacity, apparently in proportion to 
the degree of sperm 'ripeness' and thereby, of fertility (Lindahl 
et al, 1952). The light-reflecting capacity of the sperm cell may 
also be related to another optical change associated with sperm 
ripening, namely the increase in 'luminosity' of the sperm surface 
in dark-field illumination. 

It has been claimed that the spermatozoon possesses at the head 
and tail small, but directly opposite, electrical charges. However, 
all that can be claimed with certainty is that an electric charge is 
associated with the sperm cell but that its sign and magnitude depend 
largely on the concentration of the various positively and negatively 
charged ions in the surrounding medium. The following values for 
electro-conductivity in semen at 25°, expressed in reciprocal 
ohmsxlO"*, were given by Bernstein and Shergin (1936): bull 
89-5-116-3, ram 48-5-80-5, stallion 111-3-129-5, boar 123-3-134-6, 
rabbit 85-5-101 -4; and by Zagami (1939): man 88-107 (at 20°), and 
dog 129-138. 

Much scientific interest and practical importance in the rating of 
semen quality attaches to the characteristic periodic changes in 
electrical impedance which occur in semen samples with high sperm 
density and motility, and which have been shown by Rothschild 
(1948^, 1949, 1950a) to be associated closely with the so-called 
'wave motion' of the spermatozoa. When a drop of ram or bull 
semen of high density is placed on a microscope slide and examined 
at 37° under low magnification, a characteristic phenomenon can 
be observed in the form of slow, periodically appearing bands of 
high opacity or 'waves'. Measurements by means of the impedance 

42 The Biochemistry of Semen 

bridge make it possible to assess this characteristic sperm movement 
in a more objective and quantitative manner than by visual esti- 
mates; but they can only be made with semen samples which are 
sufficiently dense to show the 'wave motion'. Assays carried out 
with the electrical method in the semen of bulls at several British 
Artificial Insemination Centres revealed an interesting correlation 
of impedance change frequency with the conception rate (Bishop 
et ai, 1954). 

Distinct from the wave-motion or locomotion 'en masse' (Walton, 
1952) is the movement of individual spermatozoa which in highly 
motile semen takes the form of so-called 'forward' or 'progressive' 
motility, but which in poor specimens is confined to side-to-side 
'oscillatory' movements (Plate IV). Recently, there have been several 
attempts to replace the subjective and semi-quantitative microscope 
appraisals of motility by more clearly defined methods, of which 
those by Bosselaar and Spronk (1952) and Rothschild (1953a, b) 
deserve special mention. 

Viscosity, specific gravity, osmotic pressure, and ionic equilibrium 

The viscosity of whole semen depends largely upon the concen- 
tration of spermatozoa. Thus for example, the viscosity of bull 
semen (relative to that of pure water which is taken as unity) can 
vary from 1-76 in a specimen containing 80,000 sperm/ywl. to 10-52 
in a sample with 2,260,000 sperm //^l. (Szumowski, 1948). Seminal 
plasma itself, seldom exceeds in bulls a relative viscosity value of 2 
but higher values have been recorded in other species, especially 
those which exhibit the phenomenon of gelation. 

The average specific gravity of whole semen is 1028 in man, 
1-011 in dog, and 1-035 in bull, with fluctuations due in the first 
place to the variable ratio between sperm and seminal plasma. The 
latter is so much lighter than the spermatozoa that in practice the 
specific gravity of semen is often found to be directly proportional 
to sperm concentration. In bull semen, low specific gravity is usually 
associated with low sperm concentration and poor 'quality', whereas 
high values accompany good density and good 'quality' (Anderson, 
1946(7). This is not surprising in view of the fact that the specific 
gravity of bull seminal plasma is not greater than that of blood 
plasma, whereas the average specific gravity of bull spermatozoa 



Cinematograph of a bull spermatozoon (in semen diluted 1 : 450) moving 
forward at a speed of about 0-15 mm. /sec. Photographic plate 
exposed for 1 sec, using dark ground illumination. Mag. x 673. Only 
the projection of the movement of the sperm-head is seen, the tail 
leaving no track. As the sperm-head is shaped like an elliptical disc, 
intense light scattering occurs only when the thin edge of the head 
is visible; during 1 sec. exposure ten images of the head were recorded 
which means that it rotated or oscillated backwards and forwards 
ten times. 

(By courtesy of Lord Rothschild) 

Chemical and Physical Properties of Semen 43 

(1-28) considerably exceeds that of the erythrocytes (11) and other 
cells of the animal body. As already mentioned (p. 8), Lindahl 
and Kihlstrom (1952) believe that the wide range of variations in the 
specific gravity of ejaculated bull spermatozoa (1-240-1 -334) is due 
to the variable proportion of 'ripe' (heavier) and 'unripe' (lighter) 
sperm cells in semen. However, whereas the specific gravity of the 
sperm cell is due to the highly condensed nuclear and protoplasmic 
protein constituents, the specific gravity of the seminal plasma is the 
direct outcome of the actual osmotic pressure exerted by electrolytes 
and is thus related to the depression of the freezing point. 

Determinations of the osmotic pressure in terms of freezing point 
depression have been carried out in the semen of several species and 
the following data (in centigrade) are available: man 0-55-0-58, bull 
0-54-0-73, ram 0-55-0-70, stallion 0-58-0-62, jackass 0-56-0-62, boar 
0-59-0-63, dog 0-58-0-60, rabbit 0-55-0-59 (Slovtzov, 1916; Roem- 
mele, 1927; Milovanov, 1934; Bernstein and Shergin, 1936; Zagami, 
1940; Salisbury, Knodt and Bratton, 1948; Nishikawa and Waide, 
1951). It would seem that generally, more reliance should be placed 
on results obtained with seminal plasma than with whole semen. 
More recently, Rothschild and Barnes (1954) carried out freezing 
point determinations on forty samples of seminal plasma from ten 
bulls of different breeds, and obtained a mean value of 0-53 with a 
standard error of 0-005. 

The electrolytes in the seminal plasma are those made available 
by the secretions of the male accessory organs. Additional informa- 
tion on the content of the inorganic constituents in semen is sum- 
marized in Table 4, but again, owing to the variable composition of 
semen, the chemical data relating to whole semen must be taken with 
due reservations. 

The interrelations between the various ions in semen differ from 
those existing in blood in several respects, but most perhaps because 
of the presence of a much higher concentration of extracellular 
potassium and a correspondingly lower content of sodium. Miescher 
(1897) who was the first to examine systematically the chemical 
composition of salmon semen, found in the ash prepared from the 
seminal plasma (parts/100): 51-0 NaCl, 8-2 KCl, 140 K2SO4, and 
26-8 Na.COa. In the trout, Schlenk (1933) recorded a value of 80 mg. 
K/lOO ml. semen. In the sea-urchin {Echinus esculent us) Rothschild 


The Biochemistry of Semen 

(1948c) found 155 mg. K/lOO ml. seminal plasma. In the higher 
mammals, the content of potassium in semen may reach 400 mg./ 
100 ml. (Table 4); it is derived mainly from the seminal plasma where 
it is found at least partly, in association with citric acid (see p. 188). 

Hydrogen ion concentration and buffering capacity 

The reaction of freshly ejaculated semen is not far from neutral 
(Table 8). On standing, the semen may become alkaline at first, 
unless precautions are taken to prevent the loss of carbon dioxide, 

Table 8. Hydrogen ion concentration in semen 




7-3 -7-9 


6-4 -7-8 




6-3 -7-8 




6-2 -6-4 






6-8 -7-5 


5-9 -7-3 


6-2 -7-8 

Arbacia pimctidata 

7-6 -7-9 

Echinus escidentus 



McKenzie, Miller and Bauguess (1938) 

Anderson (1942); Hatziolos (1937) 

Laing (1945) 

Zagami (1939) 

Lambert and McKenzie (1940) 

Zagami (1939) 

Starkov (1934) 

Zagami (1939) 

Huggins, Scott and Heinen (1942) 

Zagami (1939) 

Lambert and McKenzie (1940) 

McKenzie and Berliner (1937) 

Nishikawa and Waide (1951) 

Hayashi (1945) 

Rothschild (1948c) 

but later this change is followed, at least in those specimens which 
contain fructose and a high concentration of spermatozoa, by a 
rapid decrease of pH, owing to fructolysis and accumulation of 
lactic acid. Excessive initial alkalinity of semen in some species, 
notably in bulls and rams, often accompanies low fertility, the 
alkaline reaction being associated with absence or low concen- 
tration of sperm and with a correspondingly higher proportion of 
seminal plasma. A significant negative correlation between the pH 
value and sperm density and motility has been noted frequently 
in bulls and rams (cf. Anderson, 1945); in the latter, according to 
McKenzie and Berliner (1937), normal semen is slightly acid or at 

Chemical and Physical Properties of Semen 45 

any rate never more alkaline than pH 7-3, whereas in sterile rams the 
pH value may reach 8-6. However, the rate at which acidity increases 
after ejaculation is much more significant for the assessment of 
semen than the initial pH value, because it is related directly to the 
actual glycolytic activity of the spermatozoa, and indirectly to 
sperm density and motility. In bull and ram semen this correlation 
is said to be so close that the decrease in pH on incubation can 
serve as an additional indicator of semen quality (cf. Anderson, 
1945; Laing, 1945; Reid, Ward and Salsbury, 1948Z)). As a matter 
of fact, however, the significance of this method is limited because 
as the pH value falls, the semen becomes too acid for spermatozoa 
to maintain their motility and metabolism. 

Some of the effects which are due to variations in hydrogen ion 
concentration in semen will be discussed in the next chapter; here 
we shall concern ourselves with the buffering capacity of semen 
which has been the subject of some interesting work, especially in 
bull and human semen (Shergin, 1935; Smith and Asdell, 1940; 
Easley, Mayer and Bogart, 1942; Sheldovsky, Belcher and Leven- 
stein, 1942; Willett and Salisbury, 1942; Anderson, 1946«). On the 
whole, bull semen is more highly buffered on the acid than on the 
alkaline side, and its normal buffering capacity depends mainly 
upon citrate and bicarbonate, but not phosphate. Anderson used 
107 specimens of seminal plasma from normal bulls and measured 
their buffering power by adding 005 ml. OIn-HCI to 01 5 ml. 
plasma, and determining the pH with the help of a glass electrode, 
before and after acid addition; the decrease in pH value obtained 
in this way was l-84±0038. He also investigated the relationship 
between buffering capacity and the period for which sperm motility 
of 70% and over, was maintained; semen specimens which kept up 
this motility for 24 hr. and upward, all had a good and fairly uniform 
buffering capacity (decrease in pH=l-74), but those with a smaller 
degree of motility at 24 hr. had a poorer buffering capacity (decrease 
in pH=200). 

Metabolism of semen, and its relation to sperm density and motility 

The two chief metabolic processes of semen, namely fructolysis 

and respiration, are both a direct outcome of the metabolic activity 

of the sperm cells; their rate is determined largely by the number 

46 The Biochemistry of Semen 

of spermatozoa in the semen and the degree of sperm motility 
(Mann, 1949). The more chemical aspects of semen metabolism 
will be discussed fully in conjunction with specific groups of sub- 
stances metabolized by spermatozoa, such as sugars, lipids and 
amino acids. Here, only the general outline of sperm metabolism 
will be given, in so far as it helps to bring out the relationship 
between the metabolic processes and other characteristics of semen, 
in particular, sperm concentration and motility. 


In the absence of oxygen, for example under the conditions of 
semen storage for artificial insemination, the spermatozoa rely on 
carbohydrate metabolism as the chief source of energy. Even before 
the identity of the seminal sugar was revealed, the rate of lactic acid 
production or 'glycolysis' was used as a method for semen appraisal 
(Comstock, 1939; Webster, 1939; MacLeod, 1941a, b, 1943Z); Moore 
and Mayer, 1941; Comstock, Green, Winters and Nordskog, 1943; 
Laing, 1945; Salisbury, 1946; Westgren, 1946.) However, the dis- 
covery of fructose and the work on fructolysis made available a 
chemical approach to several practical problems of male fertility 
(Mann, 19466, 1948«, b, 1949; Mann, Davies and Humphrey, 1949; 
Mann and Lutwak-Mann, \95\a, b; Mann and Parsons, 1950; 
Mann and Walton, 1953). 

The anaerobic incubation of freshly ejaculated semen is accom- 
panied by a progressive decline in the content of fructose with a 
simultaneous accumulation of lactic acid; in the presence of suitable 
buffer, the process of fructolysis in semen with good sperm motility 
can be shown to progress almost linearly until practically all of the 
sugar is used up (Fig. 8). On this basis a photometric method has 
been worked out for the measurement of sperm fructolysis, and the 
'index of fructolysis' has been defined as the amount of fructose (in 
mg.) utilized by 10^ spermatozoa in 1 hr. at 37° (Mann, 1948^, b). 
In normal bull semen, the index of fructolysis is about 1 -4-2 but it 
varies, and is significantly correlated with both the concentration 
and the motility of spermatozoa. Fructose is not utilized by either 
azoospermic semen, i.e. ejaculates devoid of sperm, or by necro- 
spermic semen, containing immotile spermatozoa. The existence 
of a positive correlation between the rate of fructolysis and the 

Chemical and Physical Properties of Semen 47 

concentration of motile spermatozoa has been amply confirmed by 
studies on both bull and human semen (Anderson, 1946b; Eichen- 
berger and Goossens, 1950; Bishop et al., 1954) but the existence of 
a similar correlation between the rate of fructolysis and fertility 

100 - 

Control with buffered semen 
inactivated by heating 

30 60 90 120 150 180 

Minutes of incubation at 37° C 

210 240 

Fig. 8. Fructolysis in bull semen (920,000 sperm/fil.) incubated at 37°; 
the disappearance of fructose was measured in (i) bull semen which 
has been diluted with half a volume 0-25m phosphate buffer pH 7-4, 
(ii) unbuffered semen, and (iii) buffered semen inactivated by heating. 

(Mann, 1948a) 

is still a matter of dispute; it has been claimed for bull semen by 
some authors (Gassner, Hill and Sulzberger, 1952) but was denied 
by others (Bishop et al., 1954). In both cases, however, the material 
used for the survey consisted mainly of bulls kept for breeding 
purposes at Artificial Insemination Centres. More insight into the 
relationship between fructolysis and fertility could probably be 
gained by studies on semen from bulls of subnormal fertility. 

48 The Biochemistry of Semen 

A lowered rate of fructolysis has been observed in certain cases 
of human subfertiHty (Davis and McCune, 1950; Birnberg, Sherber 
and Kurzrok, 1952). However, in human semen with its physio- 
logically low sperm density but high fructose content, it is actually 
more convenient to assess the rate of fructose utilization by measur- 
ing the formation of lactic acid (chemically or manometrically) 
rather than the disappearance of sugar. In any case, if one were to 
measure fructolysis in human semen by the disappearance of sugar, 
it would be essential to use a photometric method specific for 
fructose, and not base the results merely upon the changes in 'reduc- 
ing value', because human semen contains a fair amount of reducing 
substances other than carbohydrate, which represent a substantial 
proportion of the total 'reducing value' towards reagents such as 
cupric hydroxide, ferricyanide etc. Moreover, the content of these 
reducing substances often increases during the incubation of semen, 
thus rendering unreliable, not to say senseless, determinations of 
fructose based upon reduction measurements. 

Methylene-blue reduction test 

A method often used in the evaluation of semen quality is the 
'methylene-blue reduction test' which is the outcome of dehydro- 
genase activity of the semen and depends on the determination of 
the time which it takes a semen sample to decolorize a certain 
amount of methylene blue, under standard conditions of incuba- 
tion in vitro (S^rensen, 1942; Beck and Salisbury, 1943; VanDemark, 
Mercier and Salisbury, 1945; Boenner, 1947). In S0rensen's original 
method the incubation was carried out in a Thunberg tube, but in 
Russia (Milovanov and Sokolovskaya, 1947) and in France (Brochart, 
1948Z)), the test was later performed by introducing a drop of semen 
mixed with methylene blue into a capillary tube and by observing 
the decolorization in the central portion of the column; in good- 
quality bull semen, with high density and motility, decolorization 
usually takes place within less than 10 min. at 20°; if it extends 
beyond 30 min., it signifies poor semen quality. However, though 
useful, the test is of limited scope, not least because as long ago as 
1941, Lardy and Phillips showed that the reduction of methylene 
blue by sperm suspensions may be markedly delayed by a variety 
of substances including glucose, lactate and citrate. 

Chemical and Physical Properties of Semen 49 


In the presence of oxygen, semen shows a considerable respiratory 
activity which is correlated both with concentration and motility 
of spermatozoa. It is usual to express sperm respiration in terms of 
Z02, a coefficient introduced by Redenz (1933) to denote ^1. O2 taken 
up by 10^ sperm cells during 1 hr. at 37°; Z02 values reported by 
Lardy and Phillips (1943a) for bull, cock, rabbit and ram semen, are 
21, 7, 11 and 22, respectively. The use of Z02 is more convenient 
than Q02 since the latter involves centrifugation of semen and 
washing of sperm with an unphysiological fluid such as distilled 
water; the average Q02 of ram sperm based on the dry weight of 
washed sperm is about 8. Further references to the subject of sperm 
respiration measurements, classified according to species are listed 
in Table 9. 

Chang and Walton (1940) found a close relationship between 
motility and respiratory activity in ram sperm. Walton (1938) sug- 
gested measurements of oxygen uptake in bull semen as a supporting 
method for the assessment of semen quality. Walton and Edwards 
(1938) compared the breeding records of thirteen bulls, taking as a 
measure of their fertility the number of matings required to produce 
pregnancy in cows; when they analysed ten different samples of 
semen from each of these bulls, they found that there is a close cor- 
relation between the respiratory activity of semen and fertility 
assessed on the natural service records. But Ghosh, Casida and 
Lardy (1949), and Bishop et at., (1954) failed to establish a corre- 
lation between the respiratory activity of bull semen and fertility, 
as assessed on the basis of artificial insemination records. 

Further study will probably clear up these uncertainties but in 
general, when we consider the significance of sperm respiration 
measurements, there are certain points which must be taken into 
account. Unlike fructolysis, the respiratory activity of semen is not 
entirely exogenous since it involves, in addition to the oxidative 
removal of products of fructolysis (chiefly lactic acid), the endo- 
genous respiration, i.e. oxidation of some intracellular reserve 
material, most probably a lipid. Moreover, it is possible to create, 
experimentally at any rate, conditions under which sperm respira- 
tion can be dissociated from motility. For example, in the presence 

50 The Biochemistry of Semen 

Table 9. List of some references to work on sperm respiration 


Winchester and McKenzie, 1941. 


Redenz, 1933; Windstosser, 1935; Henle and Zittle, 1941, 1942; Lardy 
and Phillips, 1943a, b, 1944; Lardy, Hansen and Phillips, 1945; Tosic 
and Walton, 1950; Schultze and Mahler, 1952; Bishop and Salisbury, 
1954; Melrose and Terner, 1953; Bishop, Campbell, Hancock and 
Walton, 1954. 


Winberg, 1939; Lardy and Phillips, 1943a; Kosin, 1944. 


Ivanov, 1931; Bishop, 1942. 


Bishop, 1942. 


McLeod, 1939, 1943a, b; Shettles, 1940. 


Lardy and Phillips, 1943a; White, 1953. 


Ivanov, 1936; Comstock, 1939; Chang and Walton, 1940; Comstock, 
Green, Winters and Nordskog, 1943; Lardy, Winchester and Phillips, 
1945; Mann, 1945a; Mann and Lutwak-Mann, 1948; White, 1953. 
OYSTER, Saxostrea commercialis 
Humphrey, 1950. 


Warburg, 1915; Gray, 1928, 1931; Barron et ai, 1941, 1948, 1949; 
Hayashi, 1946; Rothschild, 1948a, c, d, 1950^/, I95\b; Spikes, 1949. 

of a suitable concentration of fluoride, one can abolish both motil- 
ity and fructolysis in ram spermatozoa without greatly suppressing 
the respiration (Mann and Lutwak-Mann, 1948) (Fig. 9). Another 
example is provided by the response of ram sperm to succinate. 
Thus, whereas the oxygen consumption of intact ram spermatozoa 
is not enhanced markedly by the addition of succinate, sperm cells 
treated with spermicidal detergents such as cetyltrimethylammonium 
bromide, 2-phenoxyethanol, sodium dodecylsulphate and similar 
surface-active agents, show in the presence of succinate a high rate 
of oxygen uptake although of course, the motility and the fructolysis 
have been completely abolished (Koefoed-Johnsen and Mann, 1954). 
The effect of fluoride on fructolysis is due chiefly to the inhibition 
of enolase; the addition of pyruvate to fluoride-treated spermatozoa 

Chemical and Physical Properties of Semen 5 1 

enables the lactic acid formation to continue (Ivanov, 1943; Lardy 
and Phillips, 1943c; Mann, \9A5b\ Melrose and Terner, 1952, 1953). 
But according to the last-named authors, washed bull spermatozoa 

1 I T- 



/ a - 
/ o 
/ 3 

/ bb 

' E 



pe - 












/ .^y 


/ ^y 


/ ^ y 


/ ^ / 



E / 

1^ / 
r^ / 

f ^-y 







Fig. 9. Effect offiiioride on the respiration and aerobic fructolysis of ram 
semen. Each manometer flask contained 0-5 ml. whole semen diluted 
with 1 -5 ml. Ringer-phosphate solution. Arrows show fructose content. 

(Mann & Luwak-Mann, 1948) 

incubated with fluoride and pyruvate under strictly anaerobic con- 
ditions, are unable to convert pyruvic acid entirely to lactic acid 
but instead, a dismutation takes place in which of two molecules 
pyruvic acid, only one is reduced to lactic acid, and the other is 
oxidized to carbon dioxide and acetic acid: 

52 The Biochemistry of Semen 

Aerobically, in the presence of both fluoride (0 02m) and pyruvate 
(001m), bull spermatozoa consume less oxygen than without 
fluoride; this low rate of oxygen uptake can be enhanced by the 
addition of 2 : 4-dinitrophenol (10~*m), which brings about a more 
complete oxidation of that fraction of pyruvate which is not reduced 
to lactic acid. Melrose and Terner (1952, 1953) claim that bull 
semen can be graded according to the respiratory response of washed 
spermatozoa to a system made up of fluoride, pyruvate and dini- 
trophenol, and that in highly fertile samples the oxygen consumption 
is low in the presence of pyruvate and fluoride, but is increased two- 
fold or more, by the addition of dinitrophenol. 

Among substances which can provide exogenous material for 
sperm respiration are glycolysable sugars and lower fatty acids such 
as lactic, pyruvic and acetic acid (Fig. 10) but the species differences 
in this respect are very marked. Thus, while in mammalian sperma- 
tozoa for example, it is possible to prolong the respiratory activity 
for a considerable length of time with glucose, fructose, mannose, 
L(+)-lactate, pyruvate, propionate, butyrate, and oxaloacetate 
(Lardy and Phillips, 1944, 1945; Mann and Lutwak-Mann, 1948; 
Humphrey and Mann, 1949; Tosic and Walton, 1950; Melrose and 
Terner, 1953), the respiration of oyster spermatozoa is increased by 
a-oxoglutarate and oxaloacetate, decreased by acetate, propionate 
and butyrate, and remains unaffected by lactate, glucose, fructose 
and mannose (Humphrey, 1950). It appears also that the mode of 
action of several organic acids on the respiration of mammalian 
spermatozoa differs fundamentally from the influence exerted by the 
same substances on the sperm of lower animals. This is illustrated 
best by the example of the peculiar response of sea-urchin sperm 
to malonate (003m), which was reported by Barron and Goldinger 
(1941/)) to increase both the O2 uptake and the aerobic CO2 output 
of sperm by as much as 200%. Succinic acid is another example of a 
substance which was found to be highly effective in the respiration 
of sea-urchin sperm (Barron and Goldinger, \94\a; Goldinger and 
Barron, 1946) but has little effect on the oxygen uptake of intact 
mammalian spermatozoa. Another characteristic difference in be- 
haviour between sea-urchin and mammalian sperm concerns the 
effect of the fatty acids on the initial rate of sperm respiration. In 
the case of sea-urchin spermatozoa, the addition of a fatty acid salt 

Chemical and Physical Properties of Semen 








-• Fructose 

o o Glucose 

X X Na-lactate 


210 240 

90 120 150 

Incubation (min.) 
Fig. 10. Effect of fructose, glucose and lactate on the respiration of washed 
ram spermatozoa (the same effect was obtained also with acetate and 
pyruvate); oxygen uptake of 3 ml. buffered suspension of 0-45x10^ 

(Mann & Lutwak-Mann, 1948) 

often produces a prompt and marked rise in the initial rate of O2 
uptake. In our experience, however, the addition of organic acid 
salts to washed suspensions of mammalian spermatozoa, does not 
lead necessarily to an actual increase in the initial O2 uptake but 
instead, these substances act by maintaining and prolonging the 
initial rate of respiration, thereby delaying the decline in Oo con- 
sumption which would set in otherwise (Mann, 1949). Furthermore, 
by adding the same substances to respiring suspensions of washed 
mammalian sperm at a stage when the respiration had already begun 
to decline, one can prevent a further deterioration in the rate of O2 


77?^ Influence of Extraneous Factors, 
Hormones, and Environmental Conditions 

Sperm inhibitors and spermicidal substances. Cliemical aspects of short- 
wave radiation. Variations in hydrogen ion concentration and tonicity. 
Influence of heat and cold; sperm vitrification and 'la vie latente'. Role of 
hormones. Sperm-egg interacting substances and chemotaxis. 'Dilution 
effect' and chemical changes associated with senescence. The use of arti- 
ficial diluents in the storage of semen. 

The list of agents, both physical and chemical, which affect sperma- 
tozoa, includes among others, changes in temperature, visible light, 
short-wave radiation, atmospheric pressure, ionic strength, and a 
host of pharmacologically active substances. The vast literature on 
the subject of sperm activation and inhibition goes back as far as 
Leeuwenhoek's observation that dilution with rain water deprives 
the canine 'animalculi' of motion, and a report by his learned friend 
Johan Ham of Arnhem, on the loss of sperm motility in a patient 
dosed with turpentine. Among Spallanzani's contributions in this 
field is the discovery that freezing in snow does not necessarily kill 
the 'spermatic vermiculi' but reduces them to a state of 'lethargy' 
from which they recover when returned to higher temperature. The 
XlXth century abounds in studies on the effect of changes in the 
medium on sperm motility and survival. Prevost and Dumas (1824) 
extended Spallanzani's observations on the lethal effect of electric 
shock and certain poisons; Donne (1837) investigated the influence 
of milk, urine, and the vaginal and cervical secretions; de Quatre- 
fages (1850, 1853) described in great detail the marked toxicity of 
copper, lead and mercuric salts. Newport (1853) studied the nar- 
cotizing influence of chloroform vapours on the amphibian sperma- 
tozoa, and concluded 'that the spermatozoon does not impregnate 
when entirely deprived of its power of motion by narcotization and 
disenabled to penetrate into the envelopes of the egg'. Both Newport 


The Influence of Extraneous Factors 55 

and de Quatrefages were fully aware of the fact that sodium chloride 
and various other sodium and potassium salts, are able to stimulate 
or inhibit sperm motility, according to concentration and specific 
experimental conditions. Their results were soon confirmed and 
extended by others, including Koelliker (1856) whose paper on 
'Physiologische Studien liber die Samenfllissigkeit' still remains the 
most comprehensive survey of its kind, and includes observations 
on spermatozoa of the bull, stallion, dog, rabbit, pigeon, frog and 
fish. Koelliker pointed out that spermatozoa rendered motionless by 
dilution with water can be revived by prompt addition of salts or 
concentrated solutions of certain organic substances such as sucrose, 
glucose, lactose, glycerol, urea and various proteins. He investi- 
gated in some detail the activating influence of blood serum, male 
accessory gland secretions, and of a variety of inorganic and organic 
substances on sperm motility. It was he who found that cyanide is 
not an inhibitor of sperm motility and established that acids are, 
on the whole, more harmful to the sperm than alkalies. 

Furthermore, Koelliker noticed that if a drop of a fairly concen- 
trated solution of potassium hydroxide is mixed with a drop of 
semen on a microscopic slide, there is usually a sudden outburst of 
activity before the spermatozoa are rendered motionless. Such a 
period of short-lived stimulation which precedes the terminal loss 
of activity, is rather characteristic of various sperm-paralysing 
agents including distilled water. Schlenk (1933) aptly named the 
phenomenon 'Todeszuckung'. Not all investigators, however, seem 
to have realized the fundamental difference between short and pro- 
longed activation phenomena, and many of them tended to confuse 
a transient increase in initial motility with the state of activity essen- 
tial for the maintenance of continuous motility and for sperm sur- 
vival. Only too often substances have been pronounced as beneficial 
to spermatozoa merely because they were observed to stimulate 
motility and metabolism, no heed being paid to the fact that this 
very stimulation may have shortened, rather than prolonged, the 
life of spermatozoa. Similarly, many a substance has been declared 
detrimental to spermatozoa solely because it appeared to reduce the 
speed of movement and metabolic rate. However, quite often the 
lowering of activity tends to prolong the life of spermatozoa, and 
favours, rather than hinders, their survival. 

56 The Biochemistry of Semen 

Sperm inhibitors and spermicidal substances 

So far, detailed studies on the mechanism which underlies the 
action of sperm inhibitors, have been relatively limited in scope and 
concerned largely with chemical compounds which affect respiration 
and glycolysis, among them cyanide, azide, dinitrophenol, and 
fluoride, or which combine with sulphydryl groups, e.g. iodoacetate, 
iodoacetamide, <?-iodosobenzoate, and /7-chloromercuribenzoate. 
Even these studies, however, have clearly indicated the existence of 
remarkable species differences in sperm behaviour. Thus for instance, 
iodoacetate which is one of the strongest inhibitors of sperm activity 
in higher animals, has a pronounced stimulating action on the 
oxygen uptake of sea-urchin spermatozoa; this peculiar effect of 
iodoacetate is shared by other sulphydryl-binding compounds, as 
well as by malonate and nitrogen mustard (Barron and Goldinger, 
\9A\b', Barron, Nelson and Ardao, 1948; Barron, Seegmiller, 
Mendes and Narahara, 1948). It is interesting and important to 
note that widely divergent results may be attained with a given 
substance according to a particular set of experimental conditions: 
rabbit spermatozoa, washed and resuspended in a sugar-free isotonic 
medium, are immobilized completely by 00001m 2 : 3 : 5-triphenyl- 
tetrazolium chloride, but a 200 times higher concentration of this 
substance is ineffective towards sperm suspended in a glucose-con- 
taining medium (Bishop and Mathews, 1952). It is equally salutary 
to bear in mind that a substance which does not increase the initial 
rate of sperm activity, may nevertheless be utilized by the sperma- 
tozoa as an essential nutrient. For instance, most of the sugars 
and fatty acids which are oxidized by spermatozoa, do not act by 
increasing the initial rate of respiration but by maintaining it. 

Surprisingly enough, many substances endowed with pronounced 
pharmocological action in the whole animal, such as the alkaloids, 
appear to exert little or no effect upon spermatozoa in vitro. Sperm 
cells are also remarkably resistant to ethanol. Ivanov (1913) observed 
excellent motility in dog sperm to which he added 2-5% ethanol, 
and he managed to obtain live and normal offspring from an animal 
inseminated with semen mixed with 10% ethanol. It may be men- 
tioned here that ethanol is one of the substances which are definitely 
known to pass into semen after ingestion by the animal (Farrell, 

The Influence of Extraneous Factors 57 

1938); sulphonamides provide a similar example (Farrell, Lyman 
and Youmans, 1938; Kuehnau, 1939; Hug, 1940). 

Marked spermicidal power is characteristic of a great many sur- 
face-active agents, quinones and heavy metal compounds. A tech- 
nique for assessing the spermicidal activity of pure substances has 
been developed by Baker (1931, 1932, 1935) whose so-called 'killing 
concentration' is the lowest one capable of killing all spermatozoa 
suspended in a buffered glucose-saline solution (Baker's solution) 
within half an hour, at body temperature, under standard conditions 
in vitro. Of the many substances examined by Baker, the most 
highly spermicidal was phenylmercuric acetate (killing concentra- 
tion 0-001%). Various quinones such as toluquinone, butylquinone, 
methoxyquinone, parabenzoquinone, ethylquinone and paraxylo- 
quinone were also strongly spermicidal. Lower down the scale were 
mercuric chloride, methoxyhydroquinone, formaldehyde, methyl- 
hydroquinone, saponin, and hexylresorcinol. A critical account of 
the existing methods for testing the efficiency of spermicidal com- 
pounds is given by Millman (1952), Davidson (1953) and Gamble 

As to the mode of action of surface-active agents, such as e.g. 
cetyltrimethylammonium bromide, cetyldimethylbenzylammonium 
chloride, /7-tri/5opropy Iphenoxypolyethoxyethanol, dodecyl sulphate, 
various condensation products of long-chain fatty alcohols with 
ethylene oxide, and the numerous other ionic and non-ionic deter- 
gents, there is some evidence to show that these substances act 
directly on the constituents of the so-called lipid capsule, i.e. the 
lipid-containing outer layer which protects the surface of the sperma- 
tozoon. The mechanism of the spermicidal activity of detergents on 
the sperm may be likened to the haemolytic action of surface-active 
compounds on the erythrocytes, or the bactericidal effects of these 
substances on various microorganisms. The changes brought about 
by detergents manifest themselves in a loss of motility and fructo lytic 
power, and in a grossly altered permeability of the sperm cells as 
indicated by the leakage of cytochrome c. In contrast to intact 
spermatozoa, the respiration of sperm cells treated with suitable 
concentrations of detergents is markedly increased by succinate 
(Koefoed-Johnsen and Mann, 1954). 

As to the action of some at any rate, of the many other 

58 The Biochemistry of Semen 

spermicidal substances, including organo-metallic compounds, there 
are indications that it is due to the blockage of vital sulphydryl 
groups in the spermatozoa. Thus for example, the immobilizing effect 
of iodoacetate on bovine spermatozoa, which has been studied by 
Lardy and Phillips (19436), is most probably due to the sulphydryl 
group-binding capacity of this substance. In a war-time study 
MacLeod (1946Z)) showed that the inhibition of the metabolism and 
motility of human spermatozoa by organic arsenicals can be over- 
come by 1:2: 3-trithiolpropane, and that the inhibitory effect of 
cupric ions can be prevented by the addition of cysteine or gluta- 
thione (MacLeod, 1951). Researches by Mann and Leone (1953) 
have shown that both motility and fructolysis are abolished in 
mammalian spermatozoa by several thiol reagents, including cupric 
ions, hydrogen peroxide and o-iodobenzoate but that ergothioneine, a 
normal constituent of boar seminal plasma, can efficiently counteract 
the paralysing action of these reagents. The oxidation of sulphydryl 
groups probably explains also the spermicidal action of hydro- 
gen peroxide. The toxicity of hydrogen peroxide to spermatozoa, 
noted already by Guenther (1907), is of particular interest since this 
substance can actually be formed under certain conditions by the 
spermatozoa themselves, in the course of their aerobic metabolism, 
and is responsible for the so-called oxygen damage which occurs 
as a result of oxygenation of semen (MacLeod, 1943^; Tosic and 
Walton, 1946o, b; VanDemark, Salisbury and Bratton, 1949). Yet 
another aspect of the damaging action of hydrogen peroxide has 
been revealed by studies on the effect of X-rays on spermatozoa. 
When sea-water heavily irradiated with X-rays was used as a diluent 
for sea-urchin semen, there was a great reduction in the survival 
period of spermatozoa and a considerable delay in the cleavage of 
the eggs fertilized with these spermatozoa. The responsible toxic 
agent present in the irradiated sea-water has been identified by 
Evans (1947) as hydrogen peroxide. It is only fair to add, however, 
that not all investigators agree with the conclusion that the toxicity 
of irradiated media is due exclusively to hydrogen peroxide. 

Chemical aspects of short-wave radiation 

The chemical changes underlying the action of X-rays on sperma- 
tozoa still remain largely unexplored, and the precise targets of this 

The Influence of Extraneous Factors 59 

and other forms of radiation in the sperm cell are by no means 
established. The amount of information on this subject, however, is 
steadily mounting, ever since Bohn's (1903) and Hertwig's (1911) 
fundamental observations on the abnormal development of sea- 
urchin and frog ova inseminated with spermatozoa previously ex- 
posed to radium emanation. The exposure of the testes to relatively 
small doses of X-ray results in sterility because of the extreme 
sensitivity of the seminiferous epithelium and complete breakdown 
of spermatogenesis; but direct irradiation of ripe, ejaculated sperma- 
tozoa, in which the nuclei are in the resting state, has usually little 
or no effect on motility, longevity, morphology or metabolism of 
sperm. Nevertheless, irradiated spermatozoa are either altogether 
infertile or, if they retain the power to penetrate the ovum and effect 
syngamy, they are incapable of inducing normal development of the 
ovum owing to damaged chromatin. This conclusion is the outcome 
of extensive investigations on irradiated spermatozoa of several 
species, including sea-urchins (Henshaw, 1940; Barron, Gasvoda 
and Flood, 1949; Blum, 1951), frogs (Bardeen, 1907; Dalcq and 
Simon, 1931; Rugh, 1939), insects (Barth, 1929; Eker, 1937), rats 
(Henson, 1942; Fogg and Cowing, 1952), mice (Snell, 1935), rabbits 
(Asdell and Warren, 1931; Amoroso and Parkes, 1947; Murphree, 
Whitaker, Wilding and Rust, 1952), and fowl (Kosin, 1944). The 
irradiation of cock semen in vitro with X-ray doses up to 10,000 r 
has been shown by Kosin to have no detectable effect on the motility, 
respiration and anaerobic glycolysis, but the fertilizing capacity of 
these spermatozoa was markedly reduced already after exposure to 
200 r, and was altogether destroyed after a dose of 5500 r. These 
results serve to underline the fact that sperm fertility may react to 
extraneous factors in a different manner from sperm motility and 
metabolism. In our experience, ram semen irradiated with 100,000 r 
and examined immediately after exposure, has normal motility, 
fructolysis and adenosine triphosphate content; evidently, the X-ray 
injury inflicted upon the spermatozoa as reflected in their diminished 
fertilizing capacity, must be the result of some other chemical change, 
possibly in the state of polymerization of the deoxyribonucleic acid 
in the sperm chromatin. 

The sensitivity of the spermatogenic tissue to short-wave radiation 
is in marked contrast to the apparent resistance of the male accessory 

60 The Biochemistry of Semen 

glands which elaborate the seminal plasma. In rats after total body 
exposure to 500 r, there was occasionally a small but transient de- 
crease in the level of fructose and citric acid secreted by the acces- 
sory organs but in surviving animals the activity was restored to 
normal within a few weeks after irradiation. This was a stage when 
the spermatozoa were mostly immotile and a large proportion of 
them severely damaged (Lutwak-Mann and Mann, 19506). 

Variations in hydrogen ion concentration and tonicity 

Hydrogen ion concentration is undoubtedly one of the most 
important factors which influence the motility, viability and meta- 
bolism of spermatozoa in all species from sea-urchin to man (Cohn, 
1917, 1918; Wolf, 1921; Gellhorn, 1920, 1927; Healy and Anderson, 
1922; Mettenleiter, 1925; Barthelemy, 1926; Yamane and Kato, 
1928; Komatsu, 1929; Schlenk, 1933; Grodzinski and Marchlewski, 
1935). Most authors agree that a value just above pH 7 provides 
the optimum for the survival of spermatozoa. Sperm respiration 
is stated to be optimal at the following pH values, boar 7 •2-7- 5, 
ram, 70-7-2, bull 6-9-70, cock 7-25, rabbit 6-8 (Winchester and 
McKenzie, 1941; Lardy and Phillips, 1943«). Below the optimum, 
motility and metabolism alike decline progressively. Alkalinity on 
the other hand, up to pH 8-5 and above, has frequently been ob- 
served to enhance the movement, particularly of human spermatozoa. 
Whereas in some species including several fishes, the spermatozoa 
are known to be extremely sensitive to changes in pH, in others, e.g. 
in the frog, certain birds and mammals, they exhibit a remarkable 
degree of resistance (Gellhorn, 1920, 1922, 1927). In the case of 
rabbit sperm partially motile spermatozoa have been found within 
the range of pH 5-0-8-8 (Cole, Waletzky and Shackelford, 1940). 
More recently Emmens (1947) has shown that even at pH 9-5-10-0 
rabbit spermatozoa retain partial motility for several hours, but 
they become immotile and die rapidly at pH values below 5-8. 
According to this author, the point at which the progressive move- 
ment is abolished and motility reduced to a condition where heads 
become completely stationary but tails still retain feeble motion, 
coincides with a state when about 50% of the sperm cells can be 
shown to be dead by the differential staining method of Lasley, 
Easley and McKenzie (1942); in this method, dead spermatozoa take 

The Influence of Extraneous Factors 61 

up eosin whereas live cells remain unstained. The time required to 
reach the 50% mortality level was stated to be 6 hr. at pH 6-4-6-5, 
29 hr. at pH 7-2-7-9, 15 hr. at pH 8-5-9-5, 7 hr. at pH 9-7-9-8, and 
4 hr. 30 min. at pH 10-2. 

However, even after spermatozoa have been rendered immotile 
by excessive acidity they can still be resuscitated by alkalinization, 
always provided that the exposure to acid has not been unduly 
long(Engelmann, 1868; Lillie, 1913, 1919; Gray, 1915; Muschat, 1926; 
Schlenk, 1933). Under conditions in vitro, the time intervals at which 
the sperm can be revived, correlate well with the mortality rate 
(Emmens, 1947); no reactivation was seen when the death rate of 
rabbit sperm exceeded 80%. 

The slowing down effect of weak acids and the reactivating in- 
fluence of weak alkalis may well be of some importance for the 
activity of spermatozoa in vivo, in the various parts of the male and 
female reproductive tract. There are, however, several other im- 
portant factors which influence sperm motility and survival in vivo, 
such as the concentration of various ions and nutrients, dilution, 
and the tension of oxygen. Thus, in the epididymis, and partly also 
in the vas deferens, the spermatozoa are immotile at a pH which 
approximates neutrahty (Lanz, 1929; Bishop and Mathews, 1952); 
here, presumably, the combination of the very low oxygen tension, 
deficiency of carbohydrate and limitation of space acquires greater 
significance than the hydrogen ion concentration. 

Tonicity as a sperm-affecting factor ranks equal in importance 
with the hydrogen ion concentration. Most investigators of semen, 
including Yamane (1920), Gellhorn (1922, 1924, 1927), and Milo- 
vanov (1934), agree that on the whole, spermatozoa seem to be 
immobilized much more readily by hypotonic than hypertonic, 
diluents. However, it must be remembered that the ultimate effect of 
tonicity depends upon certain other prevailing conditions. Thus, 
for example, Emmens (1948) who studied the motility of rabbit 
spermatozoa at various pH values with diluents of different chemical 
composition and tonicity, has shown that at pH 5 •8-6-6, the sperma- 
tozoa were more sensitive to hypotonicity than to hypertonicity, but 
in an alkaline medium the situation was reversed. In an analogous 
study of ram, bull and human spermatozoa (Blackshaw and Emmens, 
1951) it was established that at all pH levels, hypertonic solutions 

62 The Biochemistry of Semen 

were less harmful to motility than hypotonic media, and that the 
relatively slight adverse effect of hypertonicity could be diminished 
by partial replacement of sodium chloride with glucose. Further- 
more, the extent to which a hypotonic or hypertonic medium can 
affect the spermatozoa, very much depends on the degree of sperm 
dilution. Highly concentrated solutions of sodium chloride incor- 
porated in so-called salt-jellies, have been shown to possess marked 
spermicidal properties (Gamble, 1953). 

Influence of heat and cold; sperm vitrification and 'la vie latente'' 

Temperature has long been known to exert a powerful influence in 
determining the onset of spermatogenesis and breeding activity in 
animals. The effect of increased temperature on the male reproduc- 
tive organs presents many intriguing questions. Hyperpyrexia fre- 
quently causes a temporary azoospermia in man, and a hot climate 
is believed to be the principal cause of certain forms of subfertility 
among domestic animals in tropical countries. Mammalian testes 
removed from the scrotum and placed in the abdominal cavity, 
where the temperature is a few degrees higher, cease to produce 
spermatozoa; degeneration of the spermatogenic tissue sets in and 
spermatogenic function is not resumed while 'experimental crypt- 
orchidism' prevails. In the guinea-pig, a complete cessation of sper- 
matogenesis can be brought about experimentally by scrotal appli- 
cation of heat (6° above the normal body temperature) for a period 
of 10 min. (Moore, 1924, 1951). A similar effect can be produced 
in rams; semen collected from such animals a week or two later 
contains only a small number of spermatozoa, mostly dead or 
degenerate; the seminal plasma on the other hand, retains its 
normal composition or shows even a slightly elevated content of 
fructose (Glover, 1954). In bulls, heat-induced azoospermia is said 
to be associated with an increased excretion of neutral steroids in 
the urine (Meschaks, 1953). Cold, like heat, has an adverse effect on 
sperm cells in vivo. An ice-pack applied for 10 min. to the testes 
of a rabbit invariably results in disintegration of spermatozoa in 
the epididymides (Chang, 1943). 

No less dramatic but different in kind are the in vitro effects of 
heat and cold on spermatozoa. Dog and rabbit spermatozoa although 
capable of survival for several hours in vitro at 40°, soon lose their 

The Influence of Extraneous Factors 63 

motility at 45° (Amantea and Krzyszkowsky, 1921; Walton, 1930). 
Cooling to a temperature just above 0° is not harmful to ejaculated 
semen in vitro, provided however, that the temperature of the 
ejaculate has been lowered gradually, preferably by successive 
stages of 5°, with an interval of 2 hr. or so, between each. Sudden 
cooling of ejaculated semen produces so-called temperature shock 
and involves rapid and irreversible loss of motility and fertilizing 
power (Gladcinova, 1937; Chang and Walton, 1940; Easley, Mayer 
and Bogart, 1942). The decline of respiration and fructolysis in 
'temperature-shocked' samples of bull semen was shown to be cor- 
related with a proportionate increase of dead, that is eosin-staining, 
sperm cells (Hancock, 1952). 

Provided that strict precautions are observed, semen can be cooled 
well below 0° without destroying the sperm. Some of the earliest 
observations concerning the effect of low temperature on sperm 
(human, bull, stallion, and frog) were made by Spallanzani (1776, 
1799). On subjecting stallion spermatozoa 'to the cold of freezing, 
by putting the glass in which they were, among snow' he made the 
following observations: 

'The same effect was produced by snow, as by the winter's cold; 
that is, in fourteen minutes, it made the spermatozoa motionless; 
although when exposed to the heat of the atmosphere, they con- 
tinued to move seven hours and a half. But an accident that hap- 
pened in this experiment, executed during summer, afforded new 
intelligence, and divested me of a prejudice. Observing that the 
vermiculi had become motionless, I took the glass from the snow, 
and left it exposed to the air, when the heat was 27°. An hour after, 
by chance observing this semen with the microscope, I was astonished 
to find all the vermiculi reanimated, and in such a manner, as if 
they had just come from the seminal vessels. I then saw, that the 
cold had not killed them, but had reduced them to a state of com- 
plete inaction. I replaced them in the snow, and in three quarters of 
an hour took them away. These are the phenomena I observed. In 
a few minutes, their vivacity relaxed, and the diminution increased, 
until they lost the progressive motion, and retained only that of oscil- 
lation, which likewise ended in a few minutes more. Exactly the 
reverse was observed, when they passed from the cold of the snow to 
the heat of the atmosphere. The first motion that appeared, was 
that of oscillation; the body and the tail begun to vibrate languidly 

64 The Biochemistry of Semen 

from right to left; then the motion was communicated to the whole 
vermicule; and, in a short time, the progressive motion begun.' 

In the XlXth century, Prevost (1840), de Quatrefages (1853), 
Mantegazza (1866), Schenk (1870) and others, experimented with 
sperm exposed to temperatures ranging from 0° to -17°, but it 
was not until 1938 when Jahnel proved that human spermatozoa 
can resist the temperature of solid carbon dioxide (-79"), and Luyet 
and Hodapp demonstrated that frog spermatozoa can survive the 
temperature of liquid air (-192°), provided that they are mixed with 
a concentrated solution of sucrose before immersion in liquid air. 

These and subsequent studies by other authors, including Shettles 
(1940), Shaffner (1942), Hoagland and Pincus (1942) and Parkes 
(1945) have provided further strong support for the general con- 
clusion, elaborated in detail by Luyet and Gehenio (1940) in their 
treatise on Life and Death at Low Temperatures, namely that sperma- 
tozoa, not unlike certain bacteria and some flagellates, are remark- 
ably resistant to low temperatures and on vitrification pass into a 
reversible condition of complete inactivity and quiescence. This 
was described by Becquerel as ia vie latente' and has been compared 
to the behaviour of a watch which, though well wound, can be 
brought to a sudden standstill by some braking mechanism; such a 
watch will start of its own accord as soon as the brake is removed. 
The main principle underlying Luyet's thesis is that cells such as 
spermatozoa manage to survive at low temperatures if cooling is 
effected so as to by-pass the crystallization zone, by carrying the cells 
straight into the range of sub-freezing temperatures known as the 
vitrification zone, where they assume the non-crystalline, glass-like, 
or vitreous state. The passage on thawing from the vitreous state 
equally deserves attention and is best achieved by rapid warming, 
again to avoid the crystallization zone. When these precautions are 
maintained, it is possible to prevent colloidal changes ordinarily 
associated with freezing and ice-crystal formation, such as dena- 
turation and coagulation of proteins, protoplasmic precipitation, 
release of enzymes and structural disarrangement. In the opinion 
of Becquerel (1936, 1938), the principal danger to the 'latent life' 
of cells at low temperatures is the damage to cellular structure 
which occurs in the freezing zone, caused by the separation of water 

The Influence of Extraneous Factors 65 

and electrolytes from the colloidal particles. This damage can be 
substantially reduced if freezing is carried out in the presence of 
certain organic substances such as sucrose, glucose, fructose, 
glycerol, ethylene glycol, gelatin, albumin and various gums, all of 
which have been used extensively in the past in freezing and freeze- 
drying experiments on bacteria, yeasts, protozoa and various other 
cells and tissues. The application of these substances, however, to 
sperm is comparatively recent. In 1938, Luyet and Hodapp ob- 
served that frog spermatozoa fail to survive the temperature of 
liquid air, but if the cooling is carried out in the presence of 40% 
sucrose at least 20% of them revive. Shaffner, Henderson and Card 
(1941) were able to keep alive 30% of fowl spermatozoa by freezing 
them to -79°, after treatment with fructose. An attempt to use 
glycerol in connection with survival experiments on frog sperma- 
tozoa was made by Rostand (1946) but it was not until 1949 when 
the remarkable properties of glycerol were brought into prominence 
thanks to Polge, Smith and Parkes (1949) as a result of their studies 
on the low temperature resistance of glycerol-treated fowl semen. In 
fowl semen diluted with an equal volume of Ringer solution and 
vitrified at -79° for 20 min. and then rapidly thawed, there was no 
significant revival of spermatozoa. On the other hand, when the 
dilution was carried out with Ringer solution containing 40% 
glycerol, the spermatozoa resumed full motility on thawing. This 
observation was soon extended to the semen of other animals, 
including the bull (Smith and Polge, 1950). A large number of cows 
have been inseminated by Polge and Rowson (1952) with glycerol- 
treated bull semen which had been stored at -79° for periods of 
many months, and the excellent fertilizing capacity of such 'deep- 
frozen' semen was proved when pregnancy occurred in 66% of the 
inseminated animals. Glycerol-frozen and thawed human semen has 
been reported to contain motile and fertile spermatozoa (Sherman 
and Bunge, 1953; Bunge and Sherman, 1953). 

As yet, there is no adequate explanation for the effect of glycerol 
on semen. The suggestion that it acts by supporting some sort of 
residual metabolism in the frozen spermatozoa is difficult to reconcile 
with our own observation that glycerol, unlike sugars and fatty acids, 
is not oxidized by bull or ram spermatozoa. More probably, glycerol 
exerts a protective influence on spermatozoa, preventing denaturation 

66 The Biochemistry of Semen 

changes during freezing. Glycerol has long been recognized in 
protein and enzyme chemistry as a convenient, 'stabilizing' agent 
which combines the properties of a protein solvent with the ability 
to protect the protein from denaturation caused by temperature 
changes. It has been shown to prevent the heat coagulation of serum 
and egg albumin (Beilinsson, 1929), and is in use in the cold storage 
of egg yolk owing to its solubilizing action on lipoproteins (cf. 
McFarlane and Hall, 1943). Lavin, Northrop and Taylor (1935) 
used glycerol in their study of pepsin at -100°; Keilin and Hartree 
(1949) discovered that the presence of glycerol at very low tempera- 
tures intensifies the absorption spectra of haemoproteins some 
twenty-five times. This in turn, made it possible to demonstrate for 
the first time, the spectrum of cytochrome in human spermatozoa 
(Mann, 1951^). The protecting influence of glycerol on sperm col- 
loids may well be linked with the electrolyte- and water-binding 
properties of this substance. In this connection, an observation by 
Luyet deserves to be mentioned, on the existence of a definite rela- 
tionship between the water-binding capacity of different solutes 
used for vitrification, and the temperature at which devitrification 
takes place on thawing; the devitrification temperature of glycerol 
is in the neighbourhood of -70° (Miner and Dalton, 1953) which is 
below that of sucrose, fructose, gelatin, various gums and most 
other solutes. 

Role of hormones 

Among factors which influence the production of semen in man 
and animals, hormones rank paramount in importance. The forma- 
tion, output, and composition of ejaculated semen are the outcome 
of a concerted action of several endocrine organs, with the pituitary 
gland and the testicular interstitial tissue in dominant positions. 

Apart from the direct gonadotrophic activity due to the game- 
togenic and interstitial-cell stimulating hormones, the anterior lobe 
of the pituitary gland exerts an indirect influence upon the male 
reproductive organs, through interaction with the thyroid gland and 
the adrenal cortex. The anterior hypophysis itself depends on stimuli 
from the central nervous system and responds in a particularly sen- 
sitive manner to impulses transmitted through the optic nerves. 
The seasonal fluctuations in the intensity of light impulses, relayed 

The Influence of Extraneous Factors 67 

through the optic nerves, are probably responsible for the changes 
associated with the so-called male sex cycle; many lower vertebrates 
produce spermatozoa only during a brief period, once a year, and 
even among mammals, there are many species in which male sexual 
activity is restricted to definite season. Sperm survival in the epidi- 
dymis and sperm transport in the female reproductive tract, are also 
two phenomena which probably depend on pituitary function; the 
former on the normal gonadotrophic activity of the anterior lobe, 
the latter on impulses transmitted to the uterus by the oxytocic 
hormone secreted in the posterior lobe. 

The function of testicular hormone, in so far as semen is con- 
cerned, is to provide the stimulus necessary for the elaboration of 
seminal plasma and for ejaculation. The male sex hormone is 
intrinsically linked with the production of seminal plasma by the 
accessory organs; it regulates the secretory activity of the accessory 
organs, and thus determines not only the output of the seminal 
plasma as a whole, but also the relative contribution of each indi- 
vidual gland towards the ultimate make-up of semen. 

The earlier morphological studies have provided much funda- 
mental information which has helped to build up our knowledge of 
the relationship between the functional state of the male accessory 
organs and androgenic activity. These investigations have shown 
that postcastrate retrogressive changes in the gross appearance and 
in the microscopic structure of the accessory organs can be pre- 
vented, or reversed, by the administration of testicular hormone. On 
this basis several so-called 'hormone indicator tests' have been 
elaborated for the detection of male sex hormone activity (cf. Moore, 
1937; Price, 1947; Dorfman, 1950). Two such tests also involved 
semen examination: the so-called 'clotting test' was based on the 
observation that the formation of a clot in the electrically-induced 
seminal discharge of a guinea-pig depends on the presence of male 
sex hormone, and the 'spermatozoon motility test' was derived 
from the observation that in the epididymis severed from the testis, 
spermatozoa survive longer if the testis is not removed from the 
body; presumably, the testicular hormone is capable of stimulating 
the epididymal cells to secrete some substance necessary for the 
preservation of epididymal spermatozoa (Moore, 1935; Parsons, 

68 The Biochemistry of Semen 

Of recent years the introduction of biochemical methods made it 
practicable to follow up and to assess androgenic or gonadotrophic 
activity, or changes due to hormonal deficiency, by means of quanti- 
tative chemical analysis of the seminal plasma. Determinations of 
fructose, citric acid and phosphatase activity in semen provide excel- 
lent evidence of the functional state of the male accessory organs of 
reproduction. These methods which will be discussed in detail later, 
are particularly useful in studies of progressive hormonal deficiency, 
such as is brought about for instance, by defective nutrition. The 
great advantage of the chemical approach is that it enables the 
assay of accessory gland function to be carried out in live animals, 
at selected intervals, and over long periods of time. 

So far, there has been little progress in investigations concerning 
the influence of hormones on semen in vitro. Several hormones have 
been variously credited with beneficial effects upon the survival, 
motility and metabolism of sperm in vitro (cf. Tschumi, 1946), but 
in actual fact, apart from isolated observations such as those 
concerning the stimulating effect of thyroxine on spermatozoa 
(Carter, 1931, 1932; Lardy and Phillips, 1943^; Schultze and Davis, 
1948, 1949; Maqsood, 1952), the evidence at hand requires much 
strengthening before the various claims are accepted as valid. The 
same is true of studies on the content of hormones in semen itself. 
There are indications that semen contains some oestrogenic sub- 
stances (Green- Army tage et al., 1947; McCullagh and Schaffenburg, 
1951; Mukherjee et al., 1951) which is not improbable since oestro- 
genic hormones occur elsewhere in the male body, notably in the 
testis and in the urine. Diczfalusy (1954) examined by counter- 
current distribution and fluorimetric analysis an alcoholic extract 
from a litre of human semen and found in this material 10 fig. of 
oestradiol-lT/S, 30 jug. oestriol, and 60 jug. oestrone, all in a free 
non-conjugated form. 

With respect to androgens, the Dirscherl-Zilliken colour reaction 
for dehydro/56>androsterone is strongly positive in extracts from 
hydrolysed spermatozoa. According to Dirscherl and Kniichel 
(1950), the content of the colour-yielding material corresponds to 
about 5-5 mg. 'dehydroandrosterone' in 100 ml. human semen, as 
compared with 0-1 mg./lOO ml. in human urine; for bull and stallion 
semen the values are given as 4-3 and 1-6 mg./lOO ml., respectively. 

The Influence of Extraneous Factors 69 

The bulk of the androgenic material appears to occur in a bound 
form and can be set free by hydrolysis with hydrochloric acid. 

c I c 

QIX 13C \ 

18CH3 1 I leC 

C C 14C / 

/^\ /.\ / \ia/ 

Q2 QIO 8C C 

C3 5C 7C 

c c 

The tetracyclic carbon skeleton 
in androgens 









Sperm-egg interacting substances and chemotaxis 

The so-called sperm-egg interacting substances comprise a group 
of agents which have received considerable attention from pioneers 
in the field of sex physiology such as Lillie (1919) and Loeb (1913), 

70 The Biochemistry of Semen 

but have acquired even more prominence and a wider significance in 
recent times, chiefly as the outcome of investigations by the schools 
of Hartmann in Germany, Runnstrom in Sweden, and Tyler in the 
United States. So far, research on these substances has been Umited 
largely to invertebrates; it has been thoroughly reviewed by Tyler 
(1948), Bielig and von Medem (1949), Runnstrom (1949, 1951), 
Brachet (1950) and Rothschild (1951^, b) and will therefore be 
mentioned here only briefly. 

The fundamental observations on sperm-egg interacting substances 
were made by Frank Lillie who discovered that the so-called 'egg- 
water', that is sea-water with which sea-urchin eggs have remained in 
contact for a while, is enriched with some substances derived from 
the eggs, and capable of inducing the agglutination of homologous 
spermatozoa. Lillie was convinced that the sperm-agglutinating 
agent plays a significant role in fertilization and called it 'fertilizin'. 
Another effect produced by egg-water, which he noted in certain 
species of marine animals, was an increase in the motility of sperma- 
tozoa. This phenomenon he also ascribed to 'fertilizin'; the sub- 
stance in the sperm, with which fertilizin was believed to combine, 
was named 'antifertilizin'. 

The sperm-egg interacting substances, mostly studied in sea- 
urchins, are sometimes referred to as 'gamones'; those derived from 
eggs being called 'gynogamones', as opposed to 'androgamones' in 

Gynogamone I is the name given to the agent responsible for 
the activating influence of egg- water on spermatozoa. Hartmann, 
Schartau, Kuhn and Wallenfels (1939) thought that in the case of 
Arbacia pustulosa the sperm-activating substance is chemically re- 
lated to echinochrome, the pigment discovered in 1885 by MacMunn. 
They found that pure echinochrome A (2-ethyl-3 : 5 : 6 : 7 : 8-penta- 
hydroxynaphtoquinone) isolated from ripe eggs oi Arbacia pustulosa, 
exerts a stimulating effect on the movements of sea-urchin sperm, 
even in a 1 : 2,500,000,000 dilution. This claim, however, has been 
seriously challenged by Tyler (1939) and Cornman (1940, 1941). 

At this point it may be relevant to recall two observations, one 
by Clowes and Bachman (1921^, b) who noted that a sperm- 
stimulating agent can be separated from egg-water by distilla- 
tion, and the other a finding made by Carter (1931) that the 

The Influence of Extraneous Factors 71 

activation effect of egg-water on spermatozoa can be reproduced by 

Gynogamone II, also called 'isoagglutinin', is the name applied 
to the fertilizin responsible for the agglutinating action of egg-water 
upon spermatozoa of the same species. This substance originates in 
the gelatinous material or 'jelly coat' surrounding the eggs, and 
passes therefrom into the sea-water; chemically it is a mucoprotein 
which differs in composition according to species; the polysac- 
charide component has been reported to contain sulphuric acid in 
addition to galactose, fucose, glucose, or fructose (Vasseur, 1947, 
1949; Tyler, 1948; Bishop and Metz, 1952). 

The group of androgamones comprises three substances. Andro- 
gamone I, to which further reference will be made later in connec- 
tion with its alleged role as a sperm-immobilizing agent, antagonizes 
the action of gynogamone I (Hartmann, Schartau and Wallenfels, 
1940). It is a heat-stable, alcohol-soluble factor which can be ex- 
tracted from sea-urchin spermatozoa by sharp centrifugation or 
with methanol, but it is still uncertain whether or not it actually 
diffuses out of intact sperm cells. Androgamone II is the antifer- 
tilizin which reacts with the sperm-agglutinating gynagamone 11. 
It is an alcohol-insoluble, protein-like substance extracted by Hart- 
mann and his associates from sea-urchin sperm, and believed to 
function as a jelly-coat dissolving or precipitating factor. Andro- 
gamone III, also known as the 'egg-surface liquefying agent' or 
'lysin', is an alcohol-soluble substance found by Runnstrom, Lind- 
vall and Tiselius (1944) in sea-urchin and salmon spermatozoa, with 
a lytic action towards the cortical layer of eggs. It is probably a 
fatty acid; in its effect on sea-urchin eggs it resembles bee venom 
and certain detergents (Runnstrom and Lindvall, 1946). 

Much confusion in the past has been caused by conflicting reports 
which ascribed to gynogamones, apart from their activating or 
agglutinating action, also a definite attracting or chemotactic influ- 
ence on spermatozoa. It is doubtful whether in animals chemo taxis 
plays any serious role in guiding the spermatozoa to the eggs. 
Plants, on the other hand, provide several excellent examples for 
the existence of chemotaxis (Cook, 1945; Hawker, 1951). In mosses 
and ferns, the spermatozoids are well known to be attracted towards 
various substances, for example malic acid, sucrose, certain salts 

72 The Biochemistry of Semen 

and alkaloids; in a few instances, some of these substances have 
actually been claimed to occur in the archegonia (Pfeffer, 1884; 
Shibata, 1911). Bracken spermatozoa are attracted by the cis but 
not the trans, configuration of organic acids; thus, they can be 
shown to move towards maleic but not fumaric, and towards citra- 
conic but not mesaconic, acid (Rothschild, 1952). Several organic 
compounds, including some simple hydrocarbons, ethers and esters, 
have been shown to possess chemotropic activity for the sperm of 
certain Fucaceae (Cook, Elvidge and Heilbron, 1948; Cook and 
Elvidge, 1951). 

Cross-poUination between two flowering plants of Forsythia is 
brought about by an exchange, followed by enzymic breakdown, of 
two glycosides of the natural flavonol pigment quercetin, carried 
with the pollen, namely rutin (quercetin rutinoside) and quercitrin 
(quercetin rhamnoside) (Kuhn and Low, 1949; Moewus, 1950). 
Plants also provide several instances of difierential distribution of 
pigments in the male and female gametes. Among the fungi, the 
small motile male gamete of some species of Allomyces is distin- 
guished from the larger female gamete by the presence of an orange- 
coloured globule in which Emerson and Fox (1940) have found 
y-carotene along with traces of isomers. In the family Fucaceae, 
chemical resolution of the pigments from the male and female 
exudates of several species has shown that the predominant colour- 
ing matter of the orange-coloured male gametes consists of /^-caro- 
tene, whereas the olive-green pigmented eggs contain chlorophyll 
and fucoxanthin (Carter, Cross, Heilbron and Jones, 1948). The 
participation of carotenoids in the reproduction of algae is indi- 
cated by studies on the unicellular flagellate alga, Chlamydomonas, 
where picrocrocin, crocin, and cis-trans-crocQtm dimethyl esters 
have been shown to play a role in the conjugation of the male and 
female gametes as well as in sex determination (Kuhn, Moewus and 
Jerchel, 1938; Kuhn and Moewus, 1940). 

In animals, the position of carotenoids as sperm-active sub- 
stances remains uncertain, but there is the significant fact that 
remarkably large amounts of carotenoid pigments occur in male 
gonads and accessory glands of reproduction of many animals, 
including mammals (Goodwin, 1950). The importance of vitamin A 
and carotene in developing and maintaining the normal germinal 

The Influence of Extraneous Factors 73 

epithelium in bulls has been stressed repeatedly by many investi- 
gators (cf. Bratton et ai, 1948). 

'Dilution effect' and chemical changes associated with sperm senescence 

The changes which result from dilution of semen have been the 
subject of much study; two distinct lines of research are recognizable. 
The chief endeavour of the mammaUan semen investigators was to 
solve the practical problem of the composition of artificial diluents, 
whereas the workers interested in the sperm of lower forms, such 
as the sea-urchin, were trying to establish the cause, rather than the 
remedy, for the effect of dilution. It may be said at once that as yet, 
there is no perfect semen diluent, and the precise mechanism of the 
'dilution effect' still remains to be solved but much progress has 
been made in both directions. 

Some of the early experiments on semen dilution, by Koelliker 
(1856), Ankermann (1857), Engelmann (1868), and others, were 
carried out with frog spermatozoa; in semen pressed out directly 
from the frog testis, the spermatozoa were found to be motionless 
but when mixed with a few parts of water, they became intensely 
motile. This activity, however, was of short duration; it began to 
decline already after a few minutes and seldom extended beyond one 
hour. Prolonged motility was obtained when water was replaced 
with 0-25 to 0-5% NaCl solutions. In the presence of higher concen- 
trations of NaCl the spermatozoa remained motionless but could be 
'revived', even after a relatively long time, by further addition of 
water. It was also shown that the presence of oxygen is not absolutely 
essential for the motihty of frog spermatozoa. This was first demon- 
strated in 1868 by Engelmann at Utrecht who found that frog sperm 
motility could be maintained for several hours in diluted semen, in 
an atmosphere of hydrogen or carbon monoxide; however, when this 
'anaerobic' motility began to decline, it was restored by pure oxygen 
or air. 

The response of fish and sea-urchin spermatozoa to dilution with 
water or salt solutions is not very different from that of frog sperm. 
The addition of water or dilute salt solutions to trout semen pro- 
vokes a shortlived burst of activity followed by gradual exhaustion 
and death of the spermatozoa; oxygen has an activating effect on 

74 The Biochemistry of Semen 

the motility and prolongs the life of the spermatozoa (Scheuring, 
1928). Trout spermatozoa have been a favourite object for investi- 
gations on the action of various cations and anions on sperm moti- 
lity (Scheuring, 1928; Gaschott, 1928; Schlenk, 1933; Schlenk and 
Kahmann, 1938). Among the many facts brought to light by these 
investigations, the effect of potassium ions merits particular atten- 
tion. In contrast to sodium chloride, a diluent containing 01 5% 
potassium chloride was found to have no activating effect on sperm 
motility, and moreover, the addition of potassium ions to a suspen- 
sion of motile sperm in sodium chloride, rendered them motionless. 
This inhibition by potassium ions, however, was shown to be 
reversible since even after prolonged storage of sperm in the pre- 
sence of potassium ions, it was still possible to restore their motility 
by dilution with water or sodium chloride solutions. The fact that 
trout spermatozoa show great activity upon dilution with water or 
sodium chloride solution but not with trout seminal plasma, has 
been attributed by Schlenk to the high potassium content of the 
latter (80 mg./lOO ml.); in his view, the rapid increase of motility 
after dilution with water should be ascribed to the decrease of potas- 
sium concentration in the seminal plasma and the passage of potas- 
sium ions from the sperm cells into the surrounding medium. 
According to this interpretation, potassium ions fulfil a double 
function in semen: they preserve the sperm energy by inducmg 
quiescence, but at the same time, they engender, as it were, a state 
of preparedness ('Bewegungsbereitschaft'). 

Much information on the effect of dilution has been gathered 
from experiments with sea-urchin semen. The spermatozoa of sea- 
urchins, unlike those of man and higher animals, are immotile in 
the absence of oxygen (Harvey, 1930; Barron, 1932). In sea-urchin 
semen which generally has a high sperm density (there are some 
2x10^° cells /ml. in Echinus esculentus), the spermatozoa are 
motionless so long as they remain undiluted, but when shed into 
or artificially diluted with, sea-water, they become intensely motile 
and with increasing dilution their oxygen uptake rises as well (Gray, 
1928, 1931). The lack of sperm movement in undiluted sea-urchin 
semen has been regarded by Gray as the outcome of mechanical 
overcrowding; each cell exercising a restraining or allelostatic effect 
on the activity of its neighbours. Other authors believed the lack 

77?^ Influence of Extraneous Factors 75 

of movement in undiluted semen to be due not so much to mutual 
restraint, as to the presence of a specific sperm-immobilizing sub- 
stance either in the sperm or in the seminal plasma. A suggestion has 
been put forward that the main cause for the lack of motility is due 
to the sperm-paralysing influence of androgamone I. Another line 
of thought was that sperm quiescence is due to the seminal plasma 
acting by virtue of its high potassium content or, alternately, its 
low hydrogen ion concentration. 

The various theories concerned with the lack of sperm movement 
in undiluted semen have been reviewed and analysed by Rothschild 
(1948c, 1950^, \95\a,b). There is, he argues, no conclusive evidence 
that the spermatozoa remain motionless in undiluted sea-urchin 
semen because of any one factor such as allelostasis, sperm-immo- 
bilization, pH, carbon dioxide or potassium ions. It is particularly 
difficult, he points out, to reconcile the observations on the immo- 
bihzing action of seminal plasma with the fact that seminal plasma 
obtained by gentle centrifugation and used as a diluent for fresh 
semen, renders the spermatozoa as active as sea-water. He found 
that the seminal plasma of Echinus esculentiis acquires sperm- 
immobilizing properties only after prolonged centrifugation of 
semen, presumably as the result of leakage of an inhibitory sub- 
stance from the cells; the rate at which the immobilizing substance 
diffuses from the spermatozoa into the seminal plasma seems to vary 
and this probably explains the conflicting reports concerning the 
effect of centrifuged seminal plasma on sperm motility. Rothschild's 
own experiments indicate that the main cause for the lack of sperm 
movement in undiluted sea-urchin semen is deficiency of oxygen; 
in fact, spermatozoa can be mobilized even in undiluted semen by 
increased oxygen tension, and deprived of motility through oxygen 
withdrawal. According to this author, the sudden outburst of 
activity upon dilution of semen, is simply due to an improved 
access of the spermatozoa to oxygen. 

As in frog and fish, the marked motility evoked in sea-urchin 
sperm by dilution is only of limited duration and is subject to pro- 
gressive decline in spite of the presence of oxygen. If the dilution 
with sea-water is very excessive, the decline may almost immediately 
follow the onset of activity. Gray (1928, 1931) thought that the 
progressive loss of activity in dilute sperm suspensions could be 

76 The Biochemistry of Semen 

explained by spontaneous and irreversible senile decay due to the 
gradual disruption of cellular organization, exhaustion of food 
reserve, depletion of energy, and autointoxication with the reaction 
products accumulated during the period of activity. He showed that 
it is possible to delay the decline of activity by replacing the sea- 
water with egg-water; a similar effect has been later demonstrated 
with solutions of the egg-surrounding jelly. In this respect, however, 
egg-water cannot be looked upon as a very specific agent. Both 
sperm motility and respiration can be extended, for instance, by the 
addition of seminal plasma, and there are several indications that 
this is due to proteins and their breakdown products in the seminal 
plasma. Hayashi (1945, 1946) experimenting with Arbacia punctu- 
lata, demonstrated the occurrence in the seminal plasma of a non- 
dialysable constituent beneficial to the viability and fertilizing 
capacity of spermatozoa. Wicklund (1949, 1952) demonstrated a 
favourable influence of serum albumin on the fertiUzing capacity of 
sea-urchin spermatozoa; she found that the fertilizing power of 
washed or aged sperm of Psammechinus miliaris was retained much 
longer following dilution with albumin solutions than with sea- 
water. Tyler (1950) and Tyler and Atkinson (1950) found that the 
life-span of sea-urchin sperm can be considerably extended by the 
addition of certain peptides and amino acids. Tyler and Rothschild 
(1951) examined the sperm metabolism of Arbacia punctulata and 
Lytechinus in sea-water enriched with amino acids and noted that 
under such experimental conditions the initial increase of respira- 
tion characteristic of the 'dilution effect', was less pronounced but 
the subsequent decline in activity .was considerably delayed, and the 
total amount of oxygen consumed greatly increased. These facts, 
coupled with evidence of non-utilization of the added amino acids, 
indicated that the amino acids enabled the spermatozoa to make 
fuller use of their endogenous substrate, probably by inducing the 
formation of complexes with copper and other toxic heavy metals 
commonly present in sea-water. This hypothesis has gained addi- 
tional support from the results of further work on the detoxicating 
effect of metal-chelating agents such as ethylenediamine tetra- 
acetate (versene), diethyldithiocarbamate, a-benzoinoxime (cupron) 
and 8-hydroxyquinoline (Tyler, 1953). Perhaps the beneficial action 
of proteins (Metz, 1945; Wicklund, 1949) and of seminal plasma 

The Influence of Extraneous Factors 11 

(Hayashi, 1945, 1946; Chang, 1949) on spermatozoa is also, partly 
at any rate, due to similar processes. 

The mechanism of inactivation and senescence induced in sea- 
urchin spermatozoa by prolonged dilution remains obscure but there 
is no reason to suppose that it differs intrinsically from senescence 
in mammalian spermatozoa. In the latter, senescence is known to be 
associated with certain definite chemical and physical changes, such 
as oxidation of intracellular sulphydryl groups which are essential 
for normal motiUty, decrease in the content of adenosine triphos- 
phate, and increased sperm permeability, which leads to the extra- 
cellular appearance of intracellular sperm-proteins; it is also probable 
that an early change in senescence causes swelHng or some other 
degeneration in the lipoprotein-containing 'lipid capsule' which 
normally surrounds the sperm cell (see p. 126). It is by no means 
unlikely that some upset in the progress of ionic exchange reactions 
involving particularly potassium ions, is also linked with senescence. 

The effect of dilution on mammalian spermatozoa is essentially 
the same as in the sperm of lower animals. Dilution with small 
volumes of saline solution produces 'activation' or excitation, a 
phenomenon well known to Koelliker and other investigators of 
the XlXth century. The extent of this activation depends of course, 
on concomitant factors such as pH, temperature, oxygen, and the 
presence of certain substances. For example, to obtain optimal 
motility and metabolism in diluted bull sperm. Lardy and Phillips 
(1943^) advise the addition of at least 0'005m potassium and 0-012m 
magnesium, with simultaneous omission of calcium ions. The need 
for potassium ions has been re-emphasized by Blackshaw (1953). 
The inclusion of phosphate, mainly as a buffer, has been advocated 
repeatedly by several authors, even though in higher concentrations 
it depresses motility and respiration of bull sperm (Bishop and 
Salisbury, 1954). Sulphate ions have been recommended by Milo- 
vanov on the ground that they prevent the swelling of spermatozoa 
and protect the 'lipid capsule' from the action of sodium chloride. 
In the writer's laboratory, the following salt solution, similar 
in composition to the Krebs-Henseleit-Ringer solution, is used 
routinely as a diluent for ram and bull spermatozoa, 100 ml. 
0-9% NaCl, 4 ml. 1-15% KCl, 1 ml. 2-11% KH2PO4, 1 ml. 3-82% 
MgSO.-VHaO, 2 ml. of a 1-3% solution of NaHCOg saturated with 

78 The Biochemistry of Semen 

CO2. In some experiments this is supplemented with (i) 0-5 g. 
fructose/ 100 ml. ('Ringer-fructose'), (ii) 20 ml. 0-25 M-phosphate 
buffer pH 7-4, the latter made up by mixing 19 ml. of an aqueous 
solution containing 0-71 g. Na2HP04 (or 1-79 g. NasHPO^.UHaO) 
with 1 ml. N-HCl ('Ringer-phosphate') or (iii) both fructose and 
phosphate ('Ringer-fructose-phosphate'). Glass-distilled water and 
analytical grade reagents are used throughout (Mann, 1946^). 

If the dilution of mammalian sperm with saline is excessive it 
leads after a short spell of increased activity to a permanent loss 
of motility, metabolic activity and fertilizing capacity. According 
to Milovanov (1934), the resistance (R) of sperm to the immobiliz- 
ing effect of dilution with large volumes of 1% NaCl, varies in 
different animals; he believes that specimens of semen capable of 
high resistance possess at the same time high fertilizing capacity. 

The formula R=— used by Milovanov in the 'resistance test', is cal- 


culated from the volume of 1% NaCl (V) required to arrest in a 
given volume of semen (v) all progressive movement of sperm-heads. 
In bull semen the test is carried out with 002 ml. semen, at 17-24°, 
and sperm motility is assessed by microscopic examination after the 
addition of successive lots of 10 ml. 1% NaCl solution (Nagornyi 
and Smimov, 1939). The R values given by Milovanov are: bull 
300-20,000, ram 100-5000, stallion 100-1500, boar 60-1000, dog 
200-600. Some doubts however, have arisen as to the significance of 
Milovanov's test in the assessment of male fertility, as well as his 
statement that the immobilizing action is due to the toxicity of 
sodium chloride as such (Emmens and Swyer, 1947, 1948; Cheng, 
Casida and Barrett, 1949). But there is general agreement that 
excessive dilution is invariably harmful to spermatozoa. This is 
reflected, among others, in Chang's (1946) finding that a constant 
number of rabbit spermatozoa has a greater fertilizing capacity 
in a small, as opposed to a large, volume of salt diluent. Chang insem- 
inated superovulated does with saline suspensions of spermatozoa, 
removed the ova 38^2 hr. later, and counted the cleaved ova. 
Results showed that the insemination of a total of 30-40 thousand 
spermatozoa in 1 ml. 0-9^o NaCl was followed by the cleavage of 
0-6% of ova, whereas the same number of cells suspended in 0- 1 ml. 
produced cleavage in 17-42% of ova. Another observation which 

The Influence of Extraneous Factors 79 

confirms the adverse effect of dilution is, that although the addition 
of fructose to an isotonic salt diluent prolongs the metabolic activity 
of bull sperm suspensions, excessive dilution with 'Ringer-fructose' 
solution leads to a decline in the rate of fructolysis (Mann and 
Lutwak-Mann, 1948). Up to a point, the changes due to the 'dilution 
effect' resemble those produced by extensive washing of spermatozoa. 
Thus for example, whereas one careful washing of centrifuged ram 
sperm with several volumes of Ringer solution causes but negligible 
damage, repeated washing results in a progressive decline in motility 
and metabolism (Mann, 1945^; White, 1953). 

The use of artificial diluents in the storage of semen 

It has been noticed repeatedly that the deleterious effect of salt 
diluents on spermatozoa can be at least partly overcome by the 
inclusion of certain organic substances, mainly of colloidal nature. 
These observations stimulated the development of the so-called 
'media', 'pabula', 'dilutors' and 'extenders', for use in the storage 
of semen for artificial insemination. 

The use of diluents in the storage of semen has its origin in 
certain experiments made by Donne, de Quatrefages, and Koelliker, 
who examined the effect of blood plasma, milk, various proteins 
and sugars, on the spermatozoa. Early in this century, Hirokawa 
(1909), Champy (1913), and others, experimented with blood plasma 
and serum. Particularly illuminating results were obtained by 
Grodzinski and Marchlewski (1938) who stored cock semen diluted 
ten times with chicken serum at 2° for periods up to eight days, 
and found, on increasing the temperature to 37°, that the sperma- 
tozoa were motile. In addition, however, these authors also found, 
in agreement with Bernstein and Lazarev (1933), that it is advisable 
to use blood serum which has been pre-treated at 55°, as otherwise 
the spermatozoa tend to agglutinate. An agglutinating and spermi- 
cidal factor, present in fresh serum but inactivated by a few days' 
storage at 4°, or 10 minutes' heating at 56°, was found by Chang 
(1947a) in human, bovine, rabbit, guinea pig, and rat serum. It is 
mainly because of its agglutinating properties that blood serum has 
not found a wider application as a semen diluent. On the other 
hand, as a direct result of immunological studies on the reactions 
between blood serum and spermatozoa, rapid progress has been 

80 The Biochemistry of Semen 

made in studies concerning spermatozoal antigens and antibodies, 
culminating in the development of several antispermatozoal sera, 
active not only tov^ards heterologous but also homologous, sperma- 
tozoa. So far, most attempts to immunize a female against sperma- 
tozoa of her own species, as a means of 'serological contraception', 
have been failures. A wealth of data on sperm immunology and 
sperm-serum interaction can be found in articles by Metchnikoff 
(1900), Moxter (1900), Godlewski (1912), Kato (1936), Henle and 
Henle (1940), Parkes (1944), Snell (1944), Tyler (1948), Smith (1949), 
Docton et al. (1952), and Kibrick et al. (1952). 

One of the first diluents used in veterinary practice for the in- 
semination of cattle in Russia, was Milovanov's 'SGC-2 dilutor' 
(13-6 g. Na2S04, 12 g. glucose, and 5 g. Witte's peptone, in 1 1. 
water). The same investigator experimented also with so-called 
gelatinized diluents which contained apart from salts and glucose, 
some gelatin, so as to endow the fluid with a jelly-like consistency. 
One such diluent developed for the storage of bull semen, has been 
the 'GPC-3-G dilutor' (1-7 g. Na2HP04, 007g. KH0PO4, 008 g. 
Na2S04, 2-85 g. glucose, 5 00 g. gelatin, 1 1. water.) A method 
adopted by the Russian workers was to use the diluted semen for 
insemination in the form of gelatin capsules. A major advance in 
the technique of semen storage has been the introduction by 
Phillips and Lardy (1939, 1940) of the 'egg-yolk-phosphate diluent' 
which became widely established both in America and in Europe 
for the purpose of preservation, transportation and insemination of 
bull semen. It is prepared by mixing freshly separated egg yolk with 
an equal volume of phosphate buffer, pH 7-4 (2 g. NagHPOi- OHgO^ 
0-2 g. KH2PO4, made up to 100 ml. with water). For actual storage, | 
and subsequent insemination, bull semen is diluted up to 100 times, 
or more, with the egg-yolk phosphate dilutor. In addition to pre- 
serving sperm viability, the dilutor protects the spermatozoa effi- 
ciently from 'temperature shock', that is from the rapid immo- 
bilization induced by sudden cooling of semen to 5-10°, the usual 
temperature for storage of semen (Chang and Walton, 1940; Easley, 
Mayer and Bogart, 1942). The chemical nature of the protecting 
substance is unknown but there are indications that it is an acetone- 
soluble but ether-insoluble, compound (Mayer and Lasley, 1944). 

Many recommendations have been made to improve the egg-yolk- 

The Influence of Extraneous Factors 81 

buffer diluent by the inclusion of various additives such as gela- 
tine (Knoop, 1941), glycine (Knoop and Krauss, 1944; Tyler and 
Tanabe, 1952), sodium citrate (Willett and Salisbury, 1942; Salis- 
bury, Knodt and Bratton, 1948), bicarbonate and glucose (Kamp- 
schmidt, Mayer, Herman and Dickerson, 1951), dialysed yolk 
(Tosic and Walton, \9A6a) and liquid whole egg (Dunn and Bratton, 
1950); to counteract the danger from bacterial contamination, cer- 
tain antibiotics are sometimes added such as penicillin, strepto- 
mycin, polymixin, aureomycin or sulphonamide drugs. On this sub- 
ject alone there is a vast number of publications of which only a 
few can be quoted here (Knodt and Salisbury, 1946; Bay ley, Cobbs 
and Barrett, 1950; Branton, James, Patrick and Newsom, 1951; 
Foote and Bratton, 1949, 1950; Hennaux, Dimitropoulos and 
Cordiez, 1947; Pursley and Herman, 1950; VanDemark, Salisbury 
and Bratton, 1949; VanDemark, Bratton and Foote, 1950; Willett, 
1950; Dunn, Bratton and Henderson, 1953). 

Some attempts were made a while ago to replace the egg-yolk 
buffer diluent by a chemically more clearly defined, artificial 
medium. Thus, Phillips and Spitzer (1946) developed the so-called 
*L.G.B.-pabulum' which contained as essential ingredients 1-2% of 
a lipid fraction (L), made up of lipositol (an inositol-containing 
phospholipid), 0-6% glucose (G), phosphate buffer (B), pH 7-4 
(0-2% KH2PO4 and 2% Na2HP04-12H20), with 0-2% galactose, 
003% of sulphasuxidine or streptomycin, and lastly 3% gum acacia, 
added to provide 'sufficient body to prevent the settling out of sperm 
upon standing in storage'. Other substitutes for egg-yolk which 
have been suggested at various times, include milk, glycerol, paraffin, 
arachis oil and synthetic plasma-substitutes such as 'periston' 
(Laplaud, Bruneel and Galland, 1951; Koch and Robillard, 1945; 
Rostand, 1946, 1952; Asher and Kaemmerer, 1950; Thacker and 
Almquist, 1953). 

It remains for future investigations to invent an ideal diluent. 
Such a diluent would be expected to combine the following features, 
isotonicity, efficient buffering capacity, nutrient value, antibacterial 
potency, stabilizing action of a 'protective colloid', anti-oxidant 
ability, and above all, good keeping quality in a ready-to-use form. 
Furthermore, it should protect semen from the effects of sudden 
changes of temperature and preserve its full fertilizing capacity for a 

82 The Biochemistry of Semen 

reasonable period at the low temperatures of storage in vitro. There 
is also the problem as to whether semen should be stored in a 
diluted form or whether it would be better to dilute it just before 
actual use. It would appear that on the whole, under natural in vivo 
conditions, spermatozoa survive best in a highly concentrated state 
when their motility is reduced to a minimum; the prolonged life-span 
of sperm in the epididymis certainly points in that direction. Another 
even more suggestive example is the behaviour of ejaculated bat 
sperm: the density of sperm as found in the bat uterus after copula- 
tion is very high, about 6 million cells //^l. (Schwab, 1952); in this 
condition the spermatozoa seem to be largely immotile, but are 
nevertheless capable of survival for several months. Such spermato- 
zoa respond to artificial dilution by becoming intensely motile, but 
then they survive for not longer than a few days. 


Intracellular Enzymes, Metalloproteins, 

Nucleoproteins, and other Protein Constituents 

of Spermatozoa 

Mechanical separation of sperm from seminal plasma; release of intra- 
cellular proteins from damaged spermatozoa. Removal of the sperm 
nucleus from the cytoplasm. Protein-bound iron, zinc, and copper. Cyto- 
chrome. Catalase. Hyaluronidase and other 'lytic' agents. Sperm nucleo- 
proteins. Deoxyribonucleic acid. The basic nuclear proteins; protamines 
and histones. The non-basic nuclear proteins; karyogen and chromosomin. 
Keratin-like protein of the sperm membrane. 

Mechanical separation of sperm from seminal plasma; release of intra- 
cellular proteins from damaged spermatozoa 
A STUDY of the proteins present in the spermatozoa themselves calls 
for an efficient separation of the sperm from the seminal plasma, by 
centrifugation and washing. However, the spermatozoa are filiform 
structures, highly vulnerable to mechanical damage. Centrifugation, 
dilution and washing may inflict an injury upon the sperm cell which, 
even if not apparent upon ordinary microscopic examination, never- 
theless results in a leakage of certain proteins from the spermatozoa 
into the surrounding medium. Thus, for instance, cytochrome c is 
easily detached from the sperm structure as a result of cellular 
damage or prolonged storage of spermatozoa (Mann, 1951a); be- 
cause of that, the spectroscopic detection of extracellular cyto- 
chrome c provides a sensitive indicator of 'senescence' changes in 
spermatozoa. Another example is the release of hyaluronidase by 
the spermatozoa (see p. 94). At one time, this phenomenon was 
ascribed to a true secretory function of the normal sperm cells 
but more recent evidence suggests that the liberation of hyaluroni- 
dase takes place in an ageing or moribund cell population. Yet 
another phenomenon in this category is the loss of lipoprotein 
from the 'lipid capsule' of the sperm cell which may easily occur as 
7 83 

84 The Biochemistry of Semen 

a result of extensive washing. However, even if the separation of 
sperm from the seminal plasma has been carried out with due care 
and attention, there is no certainty that a loss of intracellular pro- 
tein has not been incurred. For this reason, one cannot but view 
with suspicion the results of protein analyses in sperm, if they have 
been performed with spermatozoa centrifuged at high speed, or 
washed extensively with large volumes of diluents, some of them 
anisotonic or unbuffered. 

The data at present available indicate that spermatozoa have 
a much higher concentration of proteins than the seminal plasma. 
Friedrich Miescher (1870, 1878, 1897) whose fundamental studies 
provided the earliest information on the chemical nature of some 
of the sperm proteins, was also the first to point out that in salmon, 
for instance, the high dry weight and protein content of semen was 
almost entirely due to the spermatozoa, whereas the seminal plasma 
gave practically no precipitate with 2 vol. of acidified ethanol, and 
contained no more than 0-78% dry matter, of this 0-65% mineral, 
and only 013% organic, material. However, a more recent analysis 
of Sabno fontinalis has shown a content of 1 -76% nitrogen and 
0-43% phosphorus in the seminal plasma (Felix, Fischer, Krekels 
and Mohr, 1951). Sea-urchin {Arbacla punctulata) seminal plasma 
has about 0-25% protein (Hayashi, 1945). 

Table 10. Protein composition of bull semen 
(Sarkar et al, 1947) 

In dried material, ash and lipid-free 


Seminal plasma 

Total nitrogen 





















Glutamic acid 



Protein Constituents of Spermatozoa 85 

On the mammalian side, bull semen has received much attention 
from protein analysts. Zittle and O'Dell (1941«, 6) investigated the 
nature of the sulphur in bull sperm and found that over two-thirds of 
the 1-6% S present in lipid-free dry material is accounted for by 
cystine and cysteine, and the remainder by methionine. Sarkar, 
Luecke and Duncan (1947) whose results are shown in Table 10, 
analysed separately frozen-dried bull spermatozoa (20 g. dry material 
from 100 g. fresh washed sperm) and seminal plasma (1-4 g. dry 
material from 100 ml.) for total nitrogen and amino acid content. 
The amino acids were assayed by microbiological methods in protein 
hydrolysates; however, with the exception of arginine, and to a small 
extent leucine and tryptophan, the result of the amino acid analysis 
failed to reflect the different physical character and physiological 
function of proteins in spermatozoa and seminal plasma. The con- 
spicuously high content of arginine in the spermatozoa is, of course, 
due to the presence of this amino acid in the nucleoprotein, but even 
in the seminal plasma the proportion of arginine exceeds consider- 
ably that of any other amino acid, with the possible exception of 
glutamic acid. Further data on the composition of bull sperm pro- 
tein have been presented by Porter, Shankman and Melampy (1951) 
who found in extensively washed, lipid-free and dried spermatozoa 
16-7% nitrogen; m addition to the amino acids recorded previously, 
they identified aspartic acid (5%), glycine (1-7%), proHne (3-1%), 
serine (4-5%) and tyrosine (4-3%). 

There is but little information apart from some immunological 
studies, on the chemical differences between the sperm proteins of 
various species. An early attempt in this direction was made by 
Faure-Fremiet (1913) who purified 'ascaridine', a protein peculiar 
to the testicular tissue, and probably also to spermatozoa, of 
Ascaris megalocephala; an interesing account of this and other 
unusual characteristics of Ascaris sperm is given by Panijel (1951). 

Removal of the sperm mwleus from the cytoplasm 

Special techniques are required to sever the sperm-head from the 
tail, as a preliminary to protein analysis in these two morphological 
components of the sperm cell. Miescher, who pioneered in this field, 
selected for his studies fish spermatozoa where a separation can be 
accomplished relatively easily with water or dilute organic acids, 

86 The Biochemistry of Semen 

which plasmolyse the tails (together with the middle-pieces), but not 
the heads. In this way he obtained by centrifugation two portions, a 
supernatant fluid representing the cytoplasm, and a deposit con- 
sisting of sperm-heads which could be further purified by washing 
with water. 

According to Miescher's calculations, in salmon spermatozoa the 
heads and tails contribute 76 and 24% of fresh material, and 87 and 
13% of the lipid-free material, respectively. Suspensions of fish 
sperm-heads obtained by plasmolysis, centrifugation and washing, 
are largely sperm nuclei and that is why they have been used exten- 
sively for the study of nucleoproteins by Miescher and others who 
followed in his footsteps. It is, however, rather uncertain what pro- 
portion of cytoplasm defies aqueous extraction and how much 
protein is lost from the sperm-heads in the course of washing. It is 
quite likely that losses of varying magnitude occur, which would 
account for the discrepancies in analytical results obtained by 
different authors, particularly as regards the content of cytoplasmic 
and non-basic nuclear proteins of fish spermatozoa. 

The supernatant fluid obtained by centrifugation of plasmolysed 
salmon spermatozoa was found by Miescher to be rich in soluble 
proteins and lipids. On addition of ethanol he obtained two frac- 
tions, one which was ethanol-insoluble, accounted for 41-9% of dry 
material and contained mainly protein (C 51-85, H 7-10, N 14-94, 
S 1-37), and the other ethanol-soluble, equal to 51-8% of dry 
material and made up of lecithin, fat and cholesterol. Salmon 
sperm cytoplasm is known to contain phosphatases active, amongst 
others, towards adenosine triphosphate; it is devoid of deoxynucleo- 
proteins but contains apparently some ribonucleic acid and several 
free amino acids, namely alanine, valine, isoleucine, tyrosine, aspar- 
tic acid, and glutamic acid (Felix et al., 1951). 

Mammalian spermatozoa, in contrast to those of fishes, cannot be 
plasmolysed, and their heads do not come off" in water or acid. To 
overcome this obstacle, Zittle and O'Dell (\94la, b) exposed bovine 
epididymal spermatozoa to ultrasonic waves and in this way dis- 
sociated the sperm-heads from the middle-pieces and tails. On slow 
centrifugation of the disintegrated sperm suspensions, the heads 
settled out first; at increasing speed, the middle-pieces also formed 
a sediment, leaving in the supernatant fluid most of the fragmented 

Protein Constituents of Spermatozoa 


tails. The products thus obtained were extracted with lipid solvents 
and dried. The lipid-free dry weights of heads, middle-pieces and 
tails were 51, 16, and 33%, respectively, of the whole spermatozoa. 
The content of ash, nitrogen, phosphorus, sulphur, cystine, and 
methionine in the three fractions is shown in Table 11. 

Table 11. Composition of sperm-heads, middle-pieces, and tails, 

dissociated by ultrasonic disintegration of bull spermatozoa 

(Zittle and O'Dell, 1941«) 

In dried, lipid-free material (%) 





Whole sperm 
































* Corresponding to a content of 40-5% deoxyribonucleic acid. 

In the author's experience (Mann, 1949, 19516), a relatively simple 
procedure for the disruption of ram spermatozoa is to shake them 
with fine glass beads in the mechanical disintegrator of Mickle 
(1948). Such treatment leads to fragmentation of the middle-pieces 
and tails, though not of the sperm-heads, and yields on slow centri- 
fugation a yellow-coloured, opalescent fluid which probably repre- 
sents the sperm cytoplasm. This material is very rich in enzymes; it 
contains among others, the intermediary enzymes of fructolysis, 
certain phosphatases, and the complete cytochrome-cytochrome 
oxidase system, as well as a potent succinic dehydrogenase, the 
activity of which can be demonstrated both by methylene-blue reduc- 
tion and by oxygen uptake in presence of succinate. The succinic 
dehydrogenase activity shown by disintegrated ram spermatozoa 
contrasts strikingly with the behaviour of the fresh intact sperm 
cells, the O2 consumption of which is not markedly enhanced by the 
addition of succinate. The difference in enzymic behaviour between 
the intact and disrupted sperm cells has something of a parallel in 
the activity of blood carbonic anhydrase which can be demonstrated 
much more readily in laked than in unlaked, erythrocytes (Keilin 
and Mann, 1941). 

88 The Biochemistry of Semen 

The heads of ram spermatozoa separated by the process of 
mechanical disintegration can be further freed from hpoprotein 
and from adhering particles of middle-pieces and tails by repeated 
washing and differential centrifugation. Preparations obtained in 
this way consist of sperm-heads only; they were found to contain 
3 •9-4-3% phosphorus, all of it accounted for by deoxyribonucleic acid, 
but were free from lipid and acid-soluble phosphorus compounds. 

Protein-bound iron, zinc and copper 

Zittle and Zitin (1942^) found that the total iron content of dried 
lipid-free bovine epididymal sperm is about 7 mg./lOO g., more iron 
being present in the middle-pieces and tails than in the heads. Of 
the total iron, 60% was extractable with pyrophosphate and tri- 
chloroacetic acid at 100^, and was therefore assumed to be of non- 
haematin nature; an attempt to identify haematin in the non- 
extractable portion was unsuccessful. However, with the aid of a 
spectroscopic method (Mann, 1937, 1938) designed specifically for 

Table 12. Distribution of total iron, zinc and copper in ram semen 
(Mann, 1945a) 


100 ml. semen contain 

In sperm (23 ml.; 
3-5x10" cells) 

In seminal 
plasma (77 ml.) 

Fe (mg.) 
Zn (mg.) 
Cu (mg.) 



determination of haematin (as pyridine haemochromogen) in animal 
and plant tissues, the author was able to detect readily and to deter- 
mine quantitatively haematin in bull as well as in ram spermatozoa. 
The distribution in ram semen, of total iron, and also of zinc and 
copper, is shown in Table 12 (Mann, 1945^). It can be seen that in 
the ram the concentration of these three elements is much higher 
in the spermatozoa than in the seminal plasma. With the aid of a 
Mickle disintegrator, it was possible to separate the heads from the 
tails and middle-pieces and to obtain sperm-head preparations which 
contained some iron but were completely free from haematin. On 
the other hand, the 'homogenates' from disintegrated tails and 

Protein Constituents of Spermatozoa 


middle-pieces contained a high proportion of iron in the form of 
haematin, as can be seen from Table 13. Iron as well as copper and 
zinc, present in the tail and middle-piece of ram spermatozoon, is 
largely non-dialysable. Iron occurs mainly as haematin some of 
which appears to be free and the rest protein-bound, mostly in the 
form of cytochrome. Copper belongs to a protein complex which 
readily gives up the metal on treatment with acid, thus resembling 
haemocuprein, the copper-protein isolated some time ago from 
blood cells (Mann and Keilin, 1938). Zinc also forms a complex with 
a protein but unlike the zinc-protein of blood cells it has negligible 
carbonic anhydrase activity. 

Table 13. Content of total iron, haematin, zinc, and copper ^ in 
mechanically disintegrated middle-pieces and tails of ram spermatozoa 

(Spermatozoa separated from seminal plasma by centrifugation and 
washing; mechanically disrupted in Mickle's disintegrator; sperm-heads 
removed by centrifugation. The supernatant fluid ('homogenate') which 
represents disintegrated middle-pieces and tails was analysed before and 
after dialysis. Its dry weight, expressed as mg./lOO ml. semen, was 3300 
before, and 2960 after, dialysis.) 

In the homogenate from mid-pieces and tails 

Total contents 


(mg./lOO ml. 

(mg./lOO g. 

dr. wt. of 


(mg./lOO ml. 

(mg./lOO g. 

dr. wt. of 


Total Fe 





Haematin Fe 















Human semen, in contrast to ram, has a much higher zinc 
content. The first to take notice of this were Bertrand and Vladesco 
(1921) who found 5-3-220 mg. Zn/100 ml. semen. More recently, 
the problem of seminal zinc was taken up by Mawson and Fischer 
(1953) who found that apart from the high zinc content of human 
seminal plasma which is derived from the prostatic secretion (see 
p. 19), centrifuged spermatozoa of man also carry a considerable 
zinc reserve of their own, nearly 2 mg./g. dry matter. Of this, how- 
ever, only a minute fraction is endowed with carbonic anhydrase 

90 The Biochemistry of Semen 

Sea-urchin semen has approximately the same concentration 
of copper as ram semen, with a similar distribution of the metal 
between sperm and seminal plasma (Barnes and Rothschild, 1950). 


Early investigators of semen were well aware of the fact that 
spermatozoa give a positive indophenol reaction with the 'Nadi* 
reagent, particularly marked in the regions of the acrosome and 
middle-piece (Herwerden, 1913). Ostwald (1907), Voss (1922) and 
Sereni (1929), among others, made important contributions to the 
subject of sperm indophenol oxidase and noted that the intensity of 
the reaction increased towards the final stages of sperm maturation 
and after ejaculation. 

When in 1925 Keilin discovered cytochrome and identified 
indophenol oxidase with cytochrome oxidase, he noticed that of all 
the organs of a perfused frog, the heart muscle and the testicular 
tissue exhibited the strongest absorption spectrum of cytochrome. 
A little later, Brachet (1934) reported the presence of cytochrome in 
frog sperm, and Ball and Meyerhof (1940) in sea-urchin spermatozoa. 
In spite of that, attempts by several workers to detect the spectrum 
of cytochrome in mammalian spermatozoa met with failure and the 
functioning of cytochrome in mammalian semen continued to be 
deduced only indirectly from the evidence based on the oxidation 
of succinate and phenylenediamine (Shergin, 1940; Lardy and 
Phillips, 1941c; Zittle and Zitin, 1942^; MacLeod, 1943a). This led 
to some speculation, particularly in the case of human semen, about 
the mechanism of respiration, the more so, since it has been asserted 
that the oxygen consumption of human semen is associated pre- 
dominantly with the seminal plasma and not with the spermatozoa 
themselves (MacLeod, 1941a; Ross, Miller and Kurzrok, 1941; 
Zeller, 1941). 

In an efTor^ to re-examine the whole problem, the author made a 
study of the cytochrome content of mammalian spermatozoa (Mann, 
1945a, c). With the aid of the microspectroscope, an instrument 
eminently suitable for direct observation of absorption bands in 
tissues, no difficulty was experienced in the detection of the com- 
plete cytochrome spectrum in both ram and bull semen. Human 
semen has a sperm density at least ten times lower than bull semen, 

Protein Constituents of Spermatozoa 91 

but even here, the demonstration of the absorption bands of cyto- 
chrome became possible (Mann, \95\d) by the application of the 
technique of Keilin and Hartree (1949, 1950), whereby manifold 
intensification of absorption bands is brought about by means of 
liquid air. 

Our evidence for the occurrence and active participation of the 
cytochrome system in the oxidative metabolism of mammalian 
spermatozoa can be briefly summarized as follows. Whole fresh 
semen examined a little while after ejaculation shows the diffuse 
spectrum of oxidized cytochrome as well as, weakly, the absorption 
bands of reduced cytochrome a, b, and c. However, after the addi- 
tion of a reducing agent or on anaerobic incubation of the semen, 
the bands of the reduced cytochromes become much more pro- 
nounced, the cytochrome a band being more distinct than c, and 
the latter stronger than cytochrome b. The picture is similar with 
washed sperm suspensions in fructose-Ringer-phosphate solution; 
freshly prepared sperm suspensions show mainly the spectrum of 
oxidized cytochrome, which becomes reduced in the course of 
anaerobic incubation; on aeration of the incubated suspension 
cytochrome reverts to the oxidized form. The band of cytochrome a 
can be shown to undergo a typical change under the influence of 
carbon monoxide; the reaction product thus formed in the sperm 
resembling closely the carbon monoxide compound of cytochrome 
oxidase or cytochrome a^, originally described by Keilin and 
Hartree (1939) in heart muscle preparations. Carbon monoxide, 
cyanide, azide, hydroxylamine, and other typical inhibitors of the 
cytochrome system in respiring tissues, all inhibit also sperm 

An elegant experiment on the behaviour of cytochrome in sperma- 
tozoa was performed by Rothschild (1948a, d) who demonstrated 
that the oxygen uptake of sea-urchin sperm is inhibited by carbon 
monoxide and that the inhibition can be completely reversed by 
white light but not by light of the 548 m^^ wavelength; the non- 
reversal at that particular wavelength being due to lack of absorp- 
tion by cytochrome oxidase in this region of the spectrum. By 
interposing between the source of light and the microscope a colour 
filter transmitting light of the 548 m/n wavelength, Rothschild was 
able to observe spermatozoa microscopically, in the presence of 

92 The Biochemistry of Semen 

carbon monoxide, as if they were in the dark. Under these condi- 
tions, he found that carbon monoxide depressed the respiration 
without a corresponding decrease in sperm motiUty. A similar con- 
clusion that respiration can be dissociated from motility was reached 
by Robbie (1948) from his study of the effect of cyanide on the 
spermatozoa of the sand-dollar {Echinarachnius par ma). 

The cytochrome system of sea-urchin spermatozoa includes cyto- 
chrome e and in this respect, it differs from mammalian sperm 
(Keilin and Hartree, 1949). Starfish {Asterias forbesii) spermatozoa 
on the other hand, exhibit a spectroscopic pattern of cytochrome 
very similar to sea-urchins (Borei and Metz, 1951). There is also 
some evidence that cytochrome occurs in plant sperm. This follows 
from the observation by Rothschild (1951c) that the movements of 
bracken spermatozoids {Pteridium aquilinum (L.) Kuhn) are photo- 
reversibly inhibited by carbon monoxide. 

The information gained by Zittle and Zitin (1942a) from experi- 
ments on the oxidation of /7-phenylenediamine by spermatozoa 
disintegrated by sonic treatment, coupled with earlier observations 
on the indophenol colour reaction, indicated that the cytochrome 
system is located in the cytoplasm of the middle-piece and tail, 
rather than in the sperm-head. Our own spectroscopic studies led us 
to the same conclusion; the examination of the disintegrated middle- 
pieces and tails revealed the presence of all three cytochromes with 
cytochrome a predominating; these 'homogenates' oxidized rapidly 
both /7-phenylenediamine and succinic acid, and the rate of oxygen 
consumption could be substantially increased by the addition of 
cytochrome c. 


It did not escape Miescher's notice that salmon spermatozoa differ 
from other cells by their restricted ability to decompose hydrogen per- 
oxide. Some slight catalase activity has been reported in mammaUan 
semen (Shergin, 1940) but it is questionable whether this was due 
to the spermatozoa themselves or to some accidental contamination 
of semen with blood, pus or bacteria. The deficiency of catalase in 
normal and cleanly collected bull semen is in fact, so typical that 
Blom and Christensen (1944, 1947) base on it a method for rating 
the 'hygienic quality' of bull semen; the test is carried out in Denmark 

Protein Constituents of Spermatozoa 93 

in special 'catalase tubes' in whichi hydrogen peroxide is added to 
semen and the volume of evolved oxygen recorded. In ram sperma- 
tozoa, even after mechanical disintegration, we were able to detect 
only a very weak catalase activity: an extract from 0-2 g. sperm (wet 
weight) required 20 min. at 18° to decompose a quantity of hydrogen 
peroxide which would have been decomposed in 2 min. by 0001 ml. 
blood. Sea-urchin semen on the other hand, contains much more 
catalase (Evans, 1947; Rothschild, 1948c, 1950c; Barron, Gasvoda 
and Flood, 1949; Rybak and Gustafson, 1952). 

The lack of catalase in mammalian semen explains the harmful 
effects of hydrogen peroxide and pure oxygen on spermatozoa (see 
p. 58). It is also of considerable physiological interest for another 
reason, inasmuch as the spermatozoa themselves produce hydrogen 
peroxide in vitro during the oxidation of certain amino-acids (see 
p. 117). 

Hyaliironidase and other 'lytic' agents 

The term 'hyaluronidase' in its widest sense, designates the muco- 
lytic enzyme, or rather a group of enzymes, which bring about the 
depolymerization and hydrolysis of hyaluronic acid. The muco- 
polysaccharide called hyaluronic acid is a polymer of the disac- 
charide hyalobiuronic acid which consists of A^-acetylglucosamine 
and D-glucuronic acid; its enzymic degradation, that is depoly- 
merization and hydrolysis, is believed by Meyer and his school 
(1937, 1952) to be due to the opening of the A^-acetylglucosaminidic 
bonds. Thus it should be possible to assess the activity of hyalu- 
ronidase by the determination of the reducing groups liberated by 
the enzymic process. In actual practice, however, this is only possible 
with the use of purified hyaluronidase since crude enzyme prepara- 
tions often liberate additional reducing groups through the forma- 
tion of free glucuronic acid and A^-acetylglucosamine by i^-glu- 
curonidase and /S-glucosaminidase, respectively. Apart from the 
'reductimetric' method, however, there are several other ways in 
which the activity of hyaluronidase can be measured; among those 
in use is the 'mucin clot prevention (m.c.p.) test' in which the preci- 
pitation by acetic acid of the clot-like protein-hyaluronic acid 
complex is prevented by the enzyme; the 'turbidimetric' method is 
based on the observation that purified hyaluronate at pH 4-2, gives 

94 The Biochemistry of Semen 

a fairly stable colloidal suspension with dilute serum, whereas 
depolymerized hyaluronate under identical conditions remains clear; 
the 'viscosity reduction (v.r.)' method measures the decline in vis- 
cosity caused by depolymerization; in the so-called 'Spinnbarkeit'- 
method the stringiness of hyaluronic acid is assessed by means of 
a special device, before and after enzymic treatment. Each of the 
above methods, however, is open to criticism and limited in its 
scope (cf. Lundquist, 1949a; Swyer and Emmens, 1947; Meyer and 
Rapport, 1952). 

The mammalian testis and sperm are the richest animal sources 
of hyaluronidase. The existence in testes and spermatozoa of a 
'spreading' or 'diffusing' factor which, when injected intradermally, 
increases the permeability of the skin to fluids, was established by 
Hoff'man and Duran-Reynals (1931) and McClean (1930, 1931); but 
Chain and Duthie (1939, 1940) deserve the credit for being the first 
to show that purified preparations of the testicular spreading factor 
possess strong hyaluronidase activity. Their finding was soon con- 
firmed by other workers who made several attempts to purify the 
enzyme (Hahn, 1943; Freeman, Anderson, Webster and Dorfman, 
1950; Tint and Bogash, 1950). The best preparations of bovine 
testicular hyaluronidase so far available are over ten thousand times 
more active than the testicular tissue itself, but as yet, even the most 
highly purified enzyme does not appear to be a homogeneous protein. 

Hyaluronidase originates in the seminiferous epithelium of the 
mature testis, and in semen it is associated with the spermatozoa and 
not with the seminal plasma (Werthessen, Berman, Greenburg and 
Gargill, 1945; Joel and Eichenberger, 1945; Kurzrok, Leonard and 
Conrad, 1946; Swyer, 1947^; Jacquet, Plessis and Cassou, 1951). 
The content of hyaluronidase per sperm cell is highest in rabbit and 
bull; there is less of it in human and boar sperm, very little in dog, 
and practically none in birds and reptiles. 

Although it is actually a part of the sperm cell, hyaluronidase is 
nevertheless so readily released by spermatozoa into the surround- 
ing medium that it must be assumed to be located somewhere very 
close to the cell surface, possibly on the sperm-head (Hechter and 
Hadidian, 1947; Johnston and Mixner, 1947; Perlman, Leonard 
and Kurzrok, 1948). A few hours' freezing of an aqueous sperm 
suspension at -10^, or 24 hours' standing at 0°, has been found by 

Protein Constituents of Spermatozoa 95 

Swyer (1947«, b) to be quite sufficient for hyaluronidase to pass 
completely into solution. In an isotonic medium spermatozoa also 
tend to liberate hyaluronidase but when the concentration of the 
enzyme outside the cell reaches a certain level it seems to prevent 
further leakage. Indeed, under certain experimental conditions, 
spermatozoa depleted of hyaluronidase have been found to be 
capable of reabsorbing the enzyme from a hyaluronidase-rich 
medium (Emmens and Swyer, 1948; Swyer, 1951). 

The physiological role of sperm hyaluronidase is far from clear 
at present. It may be related in some as yet unknown manner, 
to the spsrmiogenic function of the testis, but there is also soms 
indication that in certain mammals at any rate, hyaluronidase acts 
by facilitating the contact between the male and female gametes 
through a direct liquefying action on the viscous gel which cements 
the follicle cells around freshly ovulated eggs. 

Much thought has been devoted in the past to the problem of the 
participation of 'lytic' agents in the process of ovum fertilization, 
and to what at one time used to be called the 'ovulase' activity of 
spermatozoa. However, the early investigators of this problem were 
in the main concerned with lower animals. In many molluscs, 
fishes and amphibia, the unfertilized egg is normally surrounded by 
a viscous 'jelly coat' and a membrane, which the spermatozoon 
must penetrate before fertiUzation can be effected. To explain this 
process, several investigators postulated the presence of lytic agents 
in the spermatozoa, capable of mediating the fusion of the gametes, 
but there has been little evidence that these agents are in fact enzymic 
in nature, until Tyler's (1939, 1942) discovery of the 'egg-membrane 
lysin', a heat-labile protein-enzyme which he extracted from the 
sperm of two molluscs, the key-hole limpet Megathura cremilata 
and the abalone Haliotis cracherodii. With sperm extracts of these 
species, the disappearance of the egg membrane could be demon- 
strated within about 3 min., if the gelatinous coat of the egg 
was present, and in less than 30 sec, if the coat has been first 

The occurrence of similar lytic enzymes in the sperm of other 
lower animals is still under discussion (cf. Tyler, 1948; Berg, 1950; 
Runnstrom, 1951). Several lytic agents have been described in sea- 
urchin spermatozoa. One of them is the previously mentioned 

96 The Biochemistry of Semen 

androgamone III ('egg-surface liquefying agent', 'sperm lysin') dis- 
covered by Runnstrom and his co-workers and shown to be a heat- 
stable alcohol-soluble substance, probably a fatty acid (p. 71). The 
other is the protein-like jelly-coat 'dissolving' or 'precipitating' 
factor, identical with Hartmann's androgamone II; the disappearance 
of the egg-jelly under the influence of this protein-factor was origin- 
ally described by Hartmann and his colleagues in Arbacia pustulosa, 
but a similar phenomenon was later observed in other sea-urchin 
species as well (Tyler and O'Melveny, 1941; Monroy and Ruff'o, 
1947; Kraus, 1950; Vasseur, 1951; Monroy and Tosi, 1952). The 
suggestion has been put forward that the agent which helps the sea- 
urchin sperm to penetrate the jelly-coat, is a mucopolysaccharase 
similar even though not identical, with hyaluronidase. This hypo- 
thesis, however, is in want of experimental support. It is also difficult 
as yet, to assign any definite role in fertilization to the proteolytic 
gelatm-liquefying enzyme which Lundblad (1950) extracted from 
the sperm of Arbacia lixula and Paracentrotus lividus. 

In mammals, the existence of an enzymic 'cumulus-dispersing 
factor' was first brought to light by Yamane (1935), Pincus (1935) 
and Pincus and Enzmann (1936) who showed that both sperm 
suspensions and extracts from rabbit spermatozoa, brought in 
contact with unfertilized rabbit ova, can disperse within a short 
time the follicle cells of the cumulus oophorus. In 1942, McClean 
and Rowlands discovered that hyaluronidase which they obtained 
not only from testes or spermatozoa, but also from snake venom 
and bacteria, can act as a cumulus-dispersing factor by liquefying 
the viscous gel which cements the follicle cells around freshly ovu- 
lated rat ova. Similar results on the mouse were reported by Fekete 
and Duran-Reynals (1943) who also noted that the gel of the 
cumulus responds to metachromatic staining with toluidine blue 
like hyaluronic acid. 

It remains one of the unsolved mysteries in the phenomenon of 
fertilization that although the actual fertilization consists ultimately 
of the fusion of a single spermatozoon with the ovum, this can take 
place apparently only after a multitude of spermatozoa have reached 
the site of fertilization. Moreover, the denudation of the ovum from 
follicular cells has also been claimed to require the presence of 
numerous spermatozoa. According to McClean and Rowlands 

Protein Constituents of Spermatozoa 97 

(1942) they are needed to create and keep up a sufficiently high con- 
centration of hyaluronidase to permit the denudation of the egg. 
This hypothesis was put to the test by Rowlands (1944) who found 
that it is possible to increase the fertilizing capacity of a subnormal 
number of rabbit spermatozoa by the addition of hyaluronidase in 
the form of extracts from whole dilute rabbit semen. Similar results 
were reported by Leonard and Kurzrok (1945, 1946). 

In view of all this, little wonder that many investigators became 
attracted by the possibility of the therapeutic application of hyalu- 
ronidase in infertility. In fact, several enthusiastic reports appeared 
of success in human infertility of oligospermic origin, achieved by 
the addition of bovine testicular hyaluronidase to human semen. 
However, subsequent investigations failed to bear out the claim 
that hyaluronidase can enhance the fertilizing capacity of sperma- 
tozoa. According to Chang (1947Z), 1949) the earlier positive results 
obtained with extracts from whole semen should be attributed not 
to hyaluronidase but to the effect of the seminal plasma as such. 
Similarly, the concept that a high sperm concentration in the vicinity 
of the egg is needed to denude the ovum from its cumulus, has been 
questioned by Leonard, Perlman and Kurzrok (1947), Austin (1948), 
and Austin and Smiles (1948) who demonstrated clearly that sperma- 
tozoa can in fact penetrate rat ova which are still enclosed in the 
cumulus. Presumably, the hyaluronidase charge carried by the sper- 
matozoa makes it possible for the individual sperm to 'burrow' its 
way through the viscous gel which cements the follicular cells. 
This, however, need not necessarily involve the dispersion of the 
cumulus, which process is probably aided by the mechanical action 
of the ciUa or by some other tubal factor. 

Another development in the field of sperm hyaluronidase per- 
taining to the role of this enzyme in fertilization, has been the 
attempt to use certain inhibitors of hyaluronidase as systemic con- 
traceptives. Among the inhibitors of hyaluronidase, presumably 
competitive in nature, are several derivatives of hyaluronic acid 
obtained by acetylation or nitration, also heparin, and a number 
of other anticoagulants and mucopolysaccharides, including a sub- 
stance present in blood serum (Ferraro, Costa and Pelegrini, 1948; 
Hadidian and Pirie, 1948; Pincus, Pirie and Chang, 1948; Meyer 
and Rapport, 1952). Another two groups of inhibitors, some of 

98 The Biochemistry of Semen 

which act irreversibly, consist of heavy metals and quinones, includ- 
ing certain quinoid compounds derived from flavonoids (Beiler and 
Martin, 1947, 1948; Rodney et ai, 1950). Much interest was aroused 
some time ago by a report that phosphorylated hesperidine, a 
potent in vitro inhibitor of hyaluronidase, can act as an 'anti- 
fertility factor' when administered to mice and human beings; 
however, attempts to substantiate this claim have, so far, been un- 
successful (Martin and Beiler, 1952; Sieve, 1952; Martin, 1953; 
Chang and Pincus, 1953; Thompson, Sturtevant and Bird, 1953). 

Sperm nucleoproteins 

In the mature sperm cell, the sperm nucleus fills the head almost 
completely, the surrounding cytoplasm being very scanty. This 
nucleus consists of closely packed chromatin embedded in a rela- 
tively small amount of nuclear sap. The first to investigate the 
chemistry of the sperm nucleus was Miescher (1878, 1897) whose 
pioneer studies laid a foundation for the modern developments in 
the chemistry and physiology of the cell nucleus, the nucleoproteins 
and the nucleic acids. As a result of Miescher's brilliant researches, 
continued and extended by such investigators as Kossel, Schmiede- 
berg, Burian, Levene, Steudel, Lynch, Hammarsten, Rasmussen and 
Linderstr0m-Lang, and many others, it is now generally recognized 
that the chief component of sperm chromatin, one which confers 
upon the paternal (haploid) chromosomes their functions as trans- 
mitters of inheritance, is by its chemical nature, a deoxyribonucleo- 
protein^ and consists of deoxyribonucleic acid conjugated with cer- 
tain basic nuclear proteins such as protamines and histones. 

Miescher used for his work chiefly salmon spermatozoa which he 
obtained mostly by stripping the live fish. A considerable advantage 
of this method is that the material thus obtained consists entirely of 
ripe spermatozoa and is therefore of uniform composition. This 
useful material, however, is not always procurable and is sometimes 
replaced by whole excised fish testes which are less suitable as they 
may contain some immature spermatozoa even during the breeding 
season. Miescher's routine procedure was to remove first the 
sperm-tails by plasmolysis with water or weak acetic acid and then 
to treat the washed suspensions of sperm-heads with ethanol and 
ether, to remove the lipids. In order to separate sperm nucleic 

Protein Constituents of Spermatozoa 99 

acid from the nuclear protein, Miescher treated the Hpid-free 
material first with cold mineral acid (e.g. 0-25-0-5°/o hydrochloric 
acid) to remove the protein, and then with sodium hydroxide 'until 
the mixture tasted distinctly caustic to the tongue', to extract nucleic 
acid from the residue. 

The deoxyribonucleoproteins in the spermatozoa of a great many 
fishes, including salmon, belong to the group of micleoprotamines 
which can be extracted from the sperm nuclei with salt solutions. A 
convenient method for such an extraction and purification has been 
described by Pollister and Mirsky (1946). Spermatozoa of the brown 
trout, Salmo fario, were 'homogenized' with 1m solution of sodium 
chloride in a Waring mixer and the very viscous extract poured into 
six volumes of water; this caused the precipitation of the nucleo- 
protamine in the form of long strands, so fibrous that they could 
be wound around a glass rod and transferred in this way to another 
container. The fibrous material was dissolved in M-NaCl and re- 
precipitated with water, and then washed successively with 65% 
ethanol, hot 95% ethanol, and finally with ether; at this stage it 
contained about 6% phosphorus and 18% nitrogen, and consisted 
of deoxyribonucleic acid and protamine in a 6 : 4 ratio. When this 
material was dissolved in M-NaCl and dialysed against M-NaCl, the 
protamine slowly passed through the cellophane membrane leaving 
behind the solution of deoxyribonucleic acid. On pouring this solu- 
tion into five volumes of ethanol, a fibrous precipitate of the nucleic 
acid was obtained, which, after drying, had a content of 8-97% 
phosphorus and 14-47% nitrogen. This composition approaches 
the theoretical value for the sodium salt of deoxyribonucleic acid, 
9-28% phosphorus and 15-58% nitrogen. 

The removal of protamine from nucleoprotamine solutions in- 
duces no significant change in viscosity. The high viscosity of nucleo- 
protamine solutions is due entirely to the highly polymerized nucleic 
acid. Deoxyribonucleic acid prepared, for instance, from herring 
spermatozoa, has a molecular weight of 800,000; each molecule of 
it requires some 100 molecules of protamine to form a molecule 
of nucleoprotamine. According to some calculations by Felix (1952), 
a single fish sperm nucleus contains 4-5x10^ nucleoprotamine 
molecules, enough to provide about 190,000 molecules per each 
chromosome, or several thousand per each gene. 

100 The Biochemistry of Semen 

Unfortunately however, the extraction with M-NaCl is not a 
universal means for the separation of sperm nucleoproteins. In the 
key-hole limpet or freshwater clam, the sperm nucleoprotein 
resists extraction with M-NaCl, but can be brought into solution 
with a 2m salt solution, whereas no nucleoprotein can be extracted 
with NaCl of either concentration from the sperm of man, bull, 
boar or ram. Moreover, dialysis against M-NaCl or extraction with 
dilute mineral acids both prove inadequate for the removal of 
nuclear proteins from mammalian spermatozoa. In such cases, the 
separation of protein can be brought about with a chloroform- 
octanol mixture (Sevag, Lackman and Smolens, 1938), but before 
this is applied it is necessary to separate the sperm nucleus from the 
remainder of the sperm cell by ultrasonic or mechanical treatment. 

Deoxyribonucleic acid 

This when freed from nuclear protein, is composed of mono- 
nucleotides, each consisting of one molecule of phosphoric acid, 
one molecule of the sugar D(-)2-deoxyribose, and one molecule of 
a purine or pyrimidine base: adenine, guanine, cytosine or thymine. 
A small amount of yet another base, 5-methylcytosine (Wyatt, 1950, 
1951), has been found so far in the sperm deoxyribonucleic acid of 
man, bull, ram, herring and sea-urchin {Echinus esculentus), but 
probably it occurs also in other species. 

In all species, deoxyribonucleic acid is confined entirely to the 
sperm nucleus as can be demonstrated by various staining methods, 
and particularly by the 'Feulgen nucleal reaction'. This reaction was 
described by Feulgen (1914, 1917) at first as a colour test for thymo- 
nucleic acid, but later it was adapted for the staining of cell nuclei 
(Feulgen and Rosenbeck, 1924). 

With thymonucleic acid itself, the test is carried out best on a 
solution of sodium nucleinate (01 g./l ml.) prepared in a boiling 
tube on the water-bath. The solution is treated with 1 ml. 2N-H2SO4, 
and left at 100° for 3 minutes, then cooled and neutralized. When a 
drop of the hydrolysate is mixed with a few ml. of Schiff^'s fuchsin- 
sulphurous acid reagent (a 0-5% solution of fuchsin decolorized 
with SO2 and the excess of SO2 removed by suction), an intense 
purple colour develops. The chemistry of the Feulgen colour re- 
action is as yet only partly understood but is believed to involve the 

Protein Constituents of Spermatozoa 101 




HC C / H.,N— C C / OC CH 

^N^ ^NH ^N^ ^NH ^NH 

Adenine Guanine Cytosine 

(6-aminopurine) (2-amino-6-hydroxy- (2-hydroxy-6- 

purine) aminopyrimidine) 

O NH2 


/^\ ^^\ 

HN C— CH3 N C— CH3 



^NH ^NH 

Thymine 5-Methyl-cytosine 

(5-methyl-2 : 6-dihydroxy- 

following steps: acid hydrolysis which splits off purine and exposes 
deoxyribose; transformation of deoxyribose into co-la evuUnic alde- 
hyde; lastly, formation of a purple-coloured reaction product of 
this labile aldehyde with fuchsin-sulphurous acid. The mechanism 
of the Feulgen reaction as exhibited by the cell nuclei in histological 
preparations, appears to be even more involved (Danielli, 1947; 
Davidson, 1950). In cellular material, however carefully carried out, 
the Feulgen reaction cannot be expected to yield results as accurate 
and specific as the colour test with pure deoxyribonucleic acid. 
Some of the difficulties inherent in the application of the reaction 
as a staining method for sperm nuclei, have been pointed out by 
Feulgen and Rosenbeck themselves, who noted that when fresh 
smears of human semen were treated with Schiff 's fuchsin-sulphurous 
acid reagent, the sperm-heads stained rather weakly, whereas 
the middle-pieces and tails, though devoid of nuclear material, 
stained strongly. This observation was followed by a demonstration 
that the fuchsin-staining material present in the middle-piece and 
tail is a lipid, 'plasmal', which can be distinguished from the 

102 The Biochemistry of Semen 

nucleoprotein of the sperm-head by its solubiUty in ethanol. Similar 
observations with animal sperm were later reported by other investi- 
gators. There is no doubt, however, that if carried out properly and 
under conditions which eliminate interference from other fuchsin- 
staining substances, the Feulgen nucleal reaction can be made 
specific for the sperm nucleus. Several authors have stressed the 
fact that the base and the posterior region of the sperm-head stains 
particularly strongly (Marza, 1930; Wislocki, 1950; Friedlaender 
and Fraser, 1952). 

Although present in the sperm nuclei of all species, the composi- 
tion of deoxyribonucleic acid varies somewhat from one species to 
another, with regard to the proportion of the various purine and 
pyrimidine bases. But within any given species, all body cells, includ- 
ing the spermatozoa, seem to yield on purification the same nucleic 
acid, that is one with the same molar ratio of adenine, guanine, 
cytosine and thymine. In the species so far examined, the ratio of 
adenine to thymine, of guanine to cytosine, and of total purines to 
total pyrimidines, was shown to equal unity. 

The results of the analysis of bases in deoxyribonucleic acid from 
ram spermatozoa are shown in Plate V. The spermatozoa were dis- 
integrated mechanically and suspensions of washed, tail-free sperm- 
heads were prepared (Mann, \95\b). Nucleic acid was separated 
from the nuclear proteins of the sperm-heads by the chloroform- 
octanol treatment, hydrolysed with formic acid, and the liberated 
bases separated on paper chromatograms by Wyatt's method, using 
as solvent an aqueous solution containing 65% /^(^propanol and 
2N-HC1. The bases set free by acid hydrolysis and determined 
spectrophotometrically according to Markham and Smith (1949) 
were: guanine, adenine, cytosine and thymine in a molar ratio of 
0-91 : M3 : 0-86 : 110. In addition, there was a small amount of 
methylcytosine which in Plate V is only just visible as a faint ultra- 
violet-absorbing band below cytosine; the molar ratio of cytosine 
to methylcytosine was 1 : 005. 

Deoxyribonucleic acid (Na-salt) of human sperm was studied by 
Chargaff and his colleagues (Chargaff, Zamenhof and Green, 1950; 
ChargafF, 1951a, b)\ it contains 16% nitrogen and 8-9% phosphorus 
(Zamenhof, Shettles and Chargaff, 1950); the specific viscosity of a 
0-135% solution in water equals 7-0, and the sedimentation constant 




Guanine 0.9I 

Adenine |.|3 

Cytosjne 0.86 


Thymine t.lO 


Contact print, taken with ultraviolet light, of a paper chromatogram 
from the acid hydrolysate of ram sperm-heads. The figures indicate 
the molar ratios. 

Protein Constituents of Spermatozoa 1 03 

(Soo) for a 0-22% solution in 0-2M-NaCl is 5-7, a value which re- 
sembles closely that given by an undegraded specimen of calf 
thymus nucleic acid. According to Elmes, Smith and White (1952), 
the molar proportions of the purine and pyrmidine bases in deoxy- 
ribonucleic acid from human sperm and tissues are: guanine 0-92 
(standard error of observations 0-036), adenine 1-23 (0-068), cyto- 
sine 0-84 (0077), thymine 1-01 (0-09) and 5-methylcytosine 0-03. 

Table 14. Salmon sperm deoxyribonucleic acid (Chargaff et al, 1951) 

(Molar ratios between the bases.) 

Purines to pyrimidines 1-02 

Adenine to thymine 1-02 

Guanine to cytosine 1-02 

Adenine to guanine 1-43 

Thymine to cytosine 1 -43 

The analysis of the nucleic acid obtained from the sperm nucleo- 
protamine of salmon, Salmo salar (Chargaff, Lipschitz, Green and 
Hodes, 1951) gives a good illustration of the regularity in the com- 
position of deoxyribonucleic acid. The nucleoprotamine was pre- 
pared by extraction with a salt solution, the nucleic acid set free by 
chloroform-octanol treatment, and after some further purification, 
recovered as the sodium salt. Two specimens were isolated in this 
way, containing 14-3% nitrogen and 8-9% phosphorus and 14-8% 
nitrogen and 8-9% phosphorus, respectively. The ultraviolet absorp- 
tion spectrum measured in M-phosphate buffer pH 7-1, exhibited 
a maximum at 260 mi-i. The specific viscosity in distilled water at 
30-3°, was found to be 29-6 for a 0-22% solution, and 5-6 and 20 
for 0-11 and 0-055% solutions, respectively. The bases were set 
free by acid hydrolysis and analysed chromatographically and 
spectroscopically. The ratios of the purine to the pyrimidine bases, 
of adenine to thymine, and of guanine to cytosine equalled unity; 
adenine and thymine exceeded guanine and cytosine by about 40% 
(Table 14). 

Not only the composition but also the content of deoxyribonucleic 
acid in the cell nucleus appears to be fairly constant and characteris- 
tic for each animal species. The haploid nucleus of the sperm cell, 
however, differs from the diploid nuclei of the somatic cells in that 
it contains a reduced amount of chromatin and consequently it is 


The Biochemistry of Semen 

assumed to possess only one-half of the amount of deoxyribonucleic 
acid present in the somatic nuclei of a given species (Boivin, Vendrely 
and Vendrely, 1948; Vendrely and Vendrely, 1948, 1949, 1952; 
Mirsky and Ris, 1949, 1951; a review by Colette Vendrely, 1952). 
To illustrate this point, Table 1 5 gives data on the content of deoxy- 
ribonucleic acid in the sperm (haploid) nuclei and in the somatic 
(diploid or polyploid) cell nuclei of various species. The values 
range from 0- 1 1 x 10~^ mg. for the diploid nucleus of a sponge cell to 
168x10"^ for a diploid cell nucleus of Amphiuma, a urodele; the 
nucleic acid in the spermatozoa of the carp, trout, pike, tench, toad, 
cock and bull, is seen to be approximately one-half the content of 
the somatic cell nuclei. A high proportion of the data listed in 
Table 15, were obtained by means of the analytical procedures 

Table 15. Deoxyribonucleic acid content of single nuclei in 
somatic cells and spermatozoa 

(Contents expressed in mg. x 10~^ deoxyribonucleic acid per cell nucleus; 
figures in brackets refer to authors.) 


Type of cell mg. 

X 10- Vnucleus 


Orange sponge, Dysidea crawshagi 




Jelly fish, Cassiopeia 


0-33 (3) 


Sea-urchin, Arbacia 




Sea-cucumbsr, Stichopus diabole 


0-67 (5) 
0-70 (5) 
0-98 (3) 
0-90 (3) 
0-90 (3) 


Limpet, Fisswella barbadensis 
Snail, Tec tar ins muricatus 


0-50 (3) 
0-67 (3) 


Cliff crab, Plagiisia depressa 
Goose barnacle 




Sturgeon, Acipenser stiirio 
Carp, Cyprinus carpio 


3-2 (3) 
3-2 (6) 
1-6 (8) 

Protein Constituents of Spermatozoa 


Trout, Salmo ir ideas Gibb. 

Salmo fario 
Pike, Esox lucius 
Tench, Tinea tinea 






Green turtle 


Domestic fowl 







(1) Davidson et al. (1950); (2) Mirsky and Ris (1949); (3) Mirsky and 
Ris (1951); (4) Vendrely and Vendrely (1948); (5) Vendrely and Vendrely 
(1949); (6) Vendrely and Vendrely (1952); (7) Vendrely (1952); (8) Vendrely 
and Vendrely (1953). 


4-9 (6) 


2-45 (6) 


5-79 (2) 


2-67 (2) 




0-85 (6) 




0-85 (6) 


168 (3) 


15 (3) 


7-33 (2) 


3-70 (2) 


4-92 (3) 


2-34 (2) 


f2-56 (1) 
12-39 (2) 


2-54 (1) 


2-20 (1) 


2-45 (1) 

Cock sperm 



2-3 (7) 


2-1 (7) 

r6-4 (4) 


<^6-2 (2) 

l8-4 (3) 


5-9 (4) 


6-9 (4) 


6-4 (4) 

Bull sperm 

r3-3 (4) 
\2-82 (2) 


5-0 (7) 


5-2 (7) 


r6-l (7) 

15-4 (7) 


50 (7) 


5-3 (7) 


5-9 (7) 

106 The Biochemistry of Semen 

developed by Schmidt and Thannhauser (1945) and Schneider 
(1945, 1946) which are based largely on determinations of phos- 
phorus and involve the removal of (i) the 'acid-soluble phosphorus 
compounds' (extraction with cold trichloroacetic acid), and (ii) the 
phospholipids (extraction with ethanol and ether), prior to the 
analysis of nucleic acid (see also Table 16). 

The basic nuclear proteins, protamines and histones 

The proteins conjugated with deoxyribonucleic acid are of the 
basic type and have been shown to be either protamines or histones 
in most instances so far examined. Protamines have been isolated 
from fish spermatozoa of several species. Of the various protamines, 
the best known are salmine from salmon and trout sperm, and 
clupeine from herring sperm. Much less is known about the other 
protamines, such as scombrine (mackerel), cyclopterine (lump- 
sucker), esocine (pike), thynnine (tunny fish), percine (perch), 
cyprinine (carp) and st urine (sturgeon). 

Judging from the molecular weight and amino acid composition, 
salmine, clupeine and scombrine have a relatively simple structure, 
there being approximately two molecules of arginine to one molecule 
of monoamino acid. Thus, for example, the analysis of salmine 
sulphate prepared from the spermatozoa of the Spring or Chum 
salmon (Tristram, 1947, 1949) suggests a molecular weight of about 
8000, with a total of 58 amino acid residues: 40 arginine, 8 isoleu- 
cine, 2 valine, 4 proline, 3 glycine, 1 alanine and 7 serine; this sal- 
mine sulphate contains 19-85% sulphuric acid, i.e. 40 equivalents 
per molecule, sufficient to combine with all arginine residues. 
Another salmine sulphate, one prepared from the sperm of the 
Columbia River salmon (Block and Boiling, 1945) is said to con- 
tain 67 amino acid residues: 47 arginine, 1 isoleucine, 3 valine, 
6 proline, 4 alanine and 6 serine (see also: Corfield and Robson, 

The results of formol titration indicate that salmine contains one 
free amino or imino group per molecule of 8000, and the end group 
assay carried out by means of the dinitrofluorobenzene method 
(Sanger, 1952) suggests that the N-terminal position is occupied by 
the imino group of proline. In clupeine, the A^-terminal position is 
also taken up by proline, whereas at the other end of the amino acid 

Table 16. Distribution of phosphorus compounds in ram semen 

(Results based on analysis of 16-8 ml. ram semen, representing 14 
ejaculates from 7 rams; average volume of single ejaculate 1-2 ml.; 
3,050,000 spermatozoa/yal. Semen separated by centrifugation for 20 min. 
at 10,000^, into sperm, 26% v/v, and seminal plasma, 74% v/v.) 

mg. P/100 ml. semen 

In whole In sperm In seminal 

semen plasma 

Total phosphorus 328-5 186-7 141-8 

Acid-soluble phosphorus (in trichloro- 
acetic acid extract) 

Pjnorg (orthophosphate determined as 

MgNH4P04) 10-3 3-1 7-2 

Po (phosphate reacting directly with 
molybdate) 11-9 4-7 7-2 

P7 (phosphorus which appears as ortho- 
phosphate after 7 min. hydrolysis 
withN-HCl) 15-4 8-1 7-3 

P30 (phosphorus which appears as 
orthophosphate after 30 min. hydro- 
lysis with N-HCl) 15-9 8-6 7-3 

Ptot. ac. sol. (total acid-soluble phosphate 

determined after incineration) 159-4 27-4 132-0 

Patp (labile phosphate of adeno- 
sine triphosphate determined in the 
Ba-salt by the method of Parnas 
and Lutwak-Mann, 1935) 4-3 4-3 00 

Phexose (phosphate of 6-phosphohexose 
determined in the supernatant from 
Ba-ATP) 6-2 60 0-2 

Phospholipid phosphorus (extracted with 
ethanol and ether from the residue 
insoluble in trichloroacetic acid) 30-8 27-9 2-9 

Deoxyribonucleic acid-phosphorus (ex- 
tracted with KOH from the residue 
left after removal of acid-soluble P 
and phospholipid; precipitated from 
the KOH extract by acid) 1110 1110* 00 

Residual phosphorus (left after removal 
of acid-soluble compounds, phos- 
pholipids and deoxyribonucleic acid) 27-3 20-4 6-9 

* Corresponding to 3-2 x 10~^ mg. deoxyribonucleic acid/sperm cell. 


108 The Biochemistry of Semen 

chain, the C-terminal position is filled by arginyl-arginine (Dirr and 
Felix, 1932). Arginine-arginine linkages occur also with great fre- 
quency in the amino acid chain itself (Felix and Schuberth, 1942). 
The following peptides have been identified as breakdown products 
of a partial hydrolysis of clupeine: arginyl-arginine, triarginyl- 
arginine, alanyl-arginyl-arginine, seryl-arginyl-arginine, and alanyl- 
alanine; on the basis of the available evidence, Felix, Fischer and 
Krekels (1952) suggested the following sequence of proline (Prol.), 
arginine (Arg.) and monoamino acids (M.) in clupeine, 

Prol. (Arg. Arg. Arg. Arg. M. M.)^ Arg. Arg. 

Salmine and clupeine, irrespective of their origin, both contain 
arginine as the sole basic amino acid; in the corresponding nucleo- 
protamines the ratio between the arginine residues of the protamines 
and the phosphoric acid equivalents of nucleic acid is not far from 
unity, usually about 0-95 (Felix, 1951). In other protamines, on the 
other hand, e.g. percine and sturine, a certain proportion of the 
basic units is present in the form of histidine and lysine. 

On close inspection of analytical data relating to the various pro- 
tamines, there stands out a considerable degree of variability in the 
amino acid composition, even in closely related species. The situa- 
tion is even more complicated in sperm nucleoproteins which con- 
tain histones instead of protamines. The histones such as occur for 
instance, in the sperm nucleoproteins of sea-urchins {arbacine of 
Mathews, 1897) and cod-fishes (gadushistone and lotahistone) are 
characterized by a wider range of amino acids, including tyrosine. 
It is not improbable that some of the observed variations in the 
amino acid composition of protamines and histones, represent dis- 
tinct species characteristics analogous to those which are encoun- 
tered in other proteins, e.g. in the globins of various haemoglobins. 
On the other hand, however, the standards for the assessment of 
chemical purity of nuclear proteins are open to criticism, and it is un- 
certain whether the examined protamines and histones were always 
really pure. Quite likely a sperm nucleus may contain in some 
cases more than one basic protein, that is, a main protamine or 
histone, together with a smaller amount of a 'subsidiary' product 
(Stedman and Stedman, 1951). Furthermore, there is also the pos- 
sibility that some of the reported differences in the composition of 

Protein Constituents of Spermatozoa 109 

nuclear proteins are simply due to the use of material containing 
variable proportions of mature and immature spermatozoa. Sperma- 
tocytic development is well known to be associated with characteris- 
tic changes in the amino acid composition of the nuclear proteins. 
Immature spermatozoa of salmon, for instance, obtained directly 
from excised testes, contain a histone instead of the salmine. Simi- 
larly, the testes of the mackerel yield on extraction with dilute 
hydrochloric acid a histone, 'scombron', instead of the protamine 
'scombrine'. This and similar observations, prompted Kossel (1928) 
to acclaim the histones as 'intermediary stages' in the transformation 
of complex proteins into protamines. 

77?^ non-basic nuclear proteins; karyogen and chromosomin 

In addition to the basic proteins, the sperm nucleus always con- 
tains some non-basic or so-called residual proteins. In lipid-free 
preparations from salmon sperm-heads, Miescher found 19-78% 
protamine (extracted with 0-25-0-5% HCl), 2-94% acid-soluble pro- 
tein material other than protamine, 60-50% of nucleic acid (ex- 
tracted with NaOH) and 16-78% of an iron-containing residue 
which was insoluble either in acid or in alkali and which he believed 
to contain 'karyogen', the 'inner-space protein substance' {Innen- 
raumsubstanz), of the sperm nucleus. 

It was found later that the residual or non-basic nuclear proteins, 
unlike the protamines and histones, contain tryptophan as a 
characteristic component. Opinions are divided, however, on the 
problem of the actual ratio of non-basic to basic proteins in the 
sperm nucleus. According to the Stedmans (1943, 1947), the nucleo- 
protamine present in the sperm-heads of salmon accounts for no 
more than 70% of the dry, lipid-free material, whereas the remainder 
is made up largely of a non-basic protein 'chromosomin', which 
contains tryptophan.* On the other hand, PoUister and Mirsky 
(1946) state that the nucleoprotamine present in trout spermatozoa 

* The Stedmans' chromosomin must not be confused with chromosin, 
a name given by Mirsky and Pollister (1946) to a complex extracted with 
M-NaCl from isolated cell nuclei of various organs, including thymus, 
liver, spleen, pancreas, brain, frog testes and bacteria. This complex 
is composed of deoxyribonucleic acid, histone, and a tryptophan-contain- 
ing protein. 

1 10 The Biochemistry of Semen 

accounts for as much as 91% of the dry, lipid-free mass of the head 
nuclei. These authors, however, also find in the nuclei a charac- 
teristic tryptophan-containing residual protein. 

Keratin-like protein of the sperm membrane 

Within the category of 'residual' sperm proteins are also certain 
highly insoluble sulphur-rich proteins, obtained from mammalian 
spermatozoa and derived probably from the sperm membrane. The 
first mention of a 'sulphur-rich substance, containing more than 
4% S in the heads of bull spermatozoa', was made in 1878 by 
Miescher, who did not, however, investigate its origin and compo- 
sition. Green (1940) extracted ram spermatozoa successively with 
dilute acid and alkali, and obtained a residue containing 19-3% 
nitrogen and 11-4% cystine, which he believed to represent the 
sperm membrane. The possibility of a keratin-like protein present 
in the sperm membrane is strengthened by the observations of Zittle 
and O'Dell (1941a) on the solubilizing action of thioglycolic acid 
and trimethylbenzylammonium hydroxide on bull spermatozoa. In 
boar sperm, the portion which remains undissolved after prolonged 
treatment with N-NaOH, consists of 'ghost' sperm-heads which 
resemble in shape the sperm membranes (Thomas and Mayer, 1949). 
A remarkable property of the sperm membrane which can be demon- 
strated microscopically, is the extraordinary elasticity of the sperm- 
head structure; this was convincingly demonstrated in the experi- 
ments of Moench and Holt (1929-32), who were able to hook the 
head of a human spermatozoon with a microsurgical needle and 
to stretch it very considerably. 


Protein Constituents and Enzymes 
of the Seminal Plasma 

Proteoses and free amino acids. Fibrinolysin and fibrinogenase. Pepsino- 
gen. Ammonia formation. Amino acid oxidase. Seminal phosphatases; 
*acid' and 'alkaline' phosphatase; 5-nucleotidase; pyrophosphatase. 
Enzymic hydrolysis of adenosine triphosphate. 

Proteoses and free amino acids 

A DISCUSSION of the nature of extracellular protein constituents of 
semen demands the recognition of certain circumstances which are 
peculiar to the seminal plasma. An important, though sadly ne- 
glected fact is that the protein content of the seminal plasma does 
not remain constant after ejaculation but undergoes rapid changes 
of enzymic character which manifest themselves in a progressive 
decrease in the concentration of non-dialysable protein-nitrogen 
and a simultaneous accumulation of non-protein nitrogen, free 
amino acids and, at a late stage, of free ammonia. Unless this is 
fully taken into account, results of protein and amino acid analysis 
in semen are but of little significance and yield no information on the 
initial distribution of nitrogenous compounds. This applies especi- 
ally to human semen as was convincingly demonstrated by Lund- 
quist (1949c, 1952), and also by Jacobsson (1950, Fig. 11). 

Even in freshly collected seminal plasma a large proportion of 
total nitrogen is found partly as a protein-like material which passes 
readily through semi-permeable membranes but is not heat-coagu- 
lable and not precipitated by trichloroacetic acid; accordingly, this 
has been classified as propeptone, hemialbumose, primary proteose, 
and secondary proteose (thioalbumose and synalbumose) (Posner, 
1888, 1892; Marshall, 1922; Goldblatt, 1935^). In human semmal 
plasma, out of a total content of about 3-5 to 5-5 g. protein-like 
material per 100 ml., no more than 18%, usually much less, is 
coagulated by heat, and about 60% passes through cellulose 



The Biochemistry of Semen 

membranes which are impermeable to blood serum proteins 
(Huggins, Scott and Heinen, 1942). The electrophoretic pattern of 
the non-dialysable portion from five different specimens of human 
seminal plasma has been examined by Gray and Huggins (1942) who 
observed four distinct components which corresponded to serum 
albumin (17-7-22-7%), a-globulin (19-8-27-8%), i5-globulin (34-3- 
44-5%), and y-globulin (11 -4-21 0%). Ross, Moore and Miller 





"E 200 



2 I50 






^,— 0NH2-N 



20 40 60 
Minutes after ejaculation 

Fig. 1 1 . Increase of non-protein nitrogen and amino-nitrogen content in 

human semen on incubation at 37°. 

(Jacobsson, 1950) 

(1942) also carried out an electrophoretic and chemical analysis 
of human seminal plasma in which they distinguished five protein 
fractions: albumin (less than 002%); 'nucleoprotein' (less than 
004%); proteose ('Pi'), which was not heat-coagulable and passed 
through a membrane of 25 A pore diameter; two water-insoluble 
proteins ('Pg' and 'P3') and a mucoprotein ('P4') which contained 
9-3% N, 10-8% hexosamine, and gave on hydrolysis with n-HCI 
reducing substances (26-8%) but no uronic acid. The strongly 
positive periodic acid-Schifif reaction which is very characteristic 

Protein Constituents and Enzymes of Seminal Plasma 113 

for human seminal plasma, is probably due to this mucoid sub- 
stance (Wislocki, 1950). 

The proteoses in trichloroacetic acid extracts from semen, which 
one encounters not only in man, but also in the ram, bull, boar, 
and other species, occasionally interfere with chemical analyses of 
certain non-protein constituents. To overcome this difficulty, it is 
advisable to replace trichloroacetic acid with other deproteinizing 
agents such as zinc hydroxide, tungstic acid, phosphotungstic acid 
or ethanol. Another source of trouble encountered in analytical 
work with semen and due to the mucinous substance in seminal 
plasma, human in particular, is that on centrifugation the mucus 
has a tendency to form a stringy mass which firmly adheres to the 
sperm cells. Caution must therefore be exercised in attributing to 
spermatozoa as such, analytical results obtained with centrifuged 
human semen. 

Several assays of free amino acids in mammalian seminal plasma 
have been carried out, mostly however, by chromatographic or 
microbiological methods, and not by chemical isolation. A notable 
exception is the work of Wagner- Jauregg (1941) who isolated 
crystalline tyrosine from an ethanolic extract of human semen. The 
following free amino acids were found to occur in the seminal plasma 
of man, glycine, threonine, alanine, valine, leucine, isoleucine, 
cystine, proline, tyrosine, phenylalanine, lysine, arginine, aspartic 
acid and glutamic acid (Jacobsson, 1950; Lundquist, 1952). In 
bovine seminal plasma serine, glycine, alanine, aspartic acid and 
glutamic acid were found (Gassner and Hopwood, 1952), and a 
similar pattern was also observed in the seminal vesicle secretion 
and in the ampullar fluid, the latter containing in addition a trace of 
tyrosine. According to Gassner, the free amino acids in bull seminal 
plasma disappear after castration, like seminal fructose and citric 
acid, but their content is not restored by testosterone administra- 
tion; furthermore, vasectomy alone, which is without effect on the 
content of fructose, causes a disappearance of amino acids from bull 
seminal plasma. 

Free amino acids occur also in fish semen; as long ago as 1923, 
leucine, lysine and alanine have been isolated in pure form from 
protein-free extracts of herring testicles (Steudel and Suzuki, 1923). 

There are indications that the amino acids and proteoses present 

114 The Biochemistry of Semen 

in the seminal plasma may be of some importance to the sperma- 
tozoa. It may be recalled that excessive dilution of semen exerts a 
deleterious effect on spermatozoa and that this can be counteracted, 
partly at least, by the inclusion in the diluting media of amino acids 
such as glycine, alanine, valine, leucine, lysine, and glutamic acid 
(p. 76). The beneficial action of these amino acids is believed to 
depend primarily on their metal-binding capacity (Tyler and Roth- 
schild, 1951). Several other effects of amino acids have been observed 
with the sperm of lower animals. Giese and Wells (1952) found 
that glycine (005m) protected the spermatozoa of Strongylocentrotus 
piirpwatus from the detrimental effect of light. Metz and Donovan 
(1950) demonstrated that in the starfish certain amino acids promote 
the agglutination of spermatozoa by egg-water of this species; in 
the absence of these amino acids agglutination does not take place. 

Fibrinolysin and fibrinogenase 

The seminal proteoses and amino acids are presumably the pro- 
ducts of proteolytic activity which in the seminal plasma is derived 
mainly from the prostatic secretion, but partly also from the 
seminal vesicle fluid. The two powerful proteolytic agents of the 
prostatic secretion are 'fibrinolysin' and 'fibrinogenase' (see also 
pp. 17 and 29). 

The coagulation of human semen is followed by liquefaction, a 
process which is catalysed by a proteolytic agent present in the 
prostatic secretion. Its discoverers, Huggins and Neal (1942) named 
it fibrinolysin because of its ability to digest blood fibrin, and its 
resemblance to the fibrinolytic agent in haemolytic streptococci 
(Tillet and Garner, 1933). However, the fibrinolytic system present 
in blood has now been resolved into several distinct components, 
whereas the streptococcal fibrinolysin has been defined as a kinase, 
i.e. an activator of the fibrinolytic enzyme preformed in the blood. 
Consequently, the name 'fibrinolysin' has been abandoned with 
reference to the streptococcal agent in favour of 'streptokinase'. 
Furthermore, it proved impossible to replace streptokinase by pro- 
static fibrinolysin as an activator of the blood enzyme (Oettle, 

The fibrinolytic activity can be assayed in human semen by the 
method of Harvey (1949), which consists in mixing a constant volume 

Protein Constituents and Enzymes of Seminal Plasma 1 1 5 

of oxalated blood plasma with varying volumes of semen, inducing 
clotting by the addition of 1-5% calcium chloride, and noting the 
time required for the clot to liquefy. Owing to the inhibitory effect 
of blood plasma on fibrinolysis, the plasma must not constitute 
more than one-tenth of the reacting system. Harvey states that the 
degree of fibrinolytic activity in semen varies with the individual 
but she found no correlation between this activity and either the 
volume of ejaculates or any characteristics of spermatozoa. Simi- 
larly, there was no positive relationship between the lysin content 
and semen viscosity. However, specimens which were exceptionally 
viscous, usually also had low fibrinolytic power. 

The precise nature of seminal fibrinolysin and its relation to 
plasmin, the fibrin-splitting agent of the blood, will not be known 
until the enzyme has been purified. Moreover, experiments by Kaulla 
and Shettles (1953) indicate that in addition to the plasmin-like 
enzyme proper, the human seminal plasma contains at least three 
other agents, (i) fibrinolysokinase, an activator of blood profibrinoly- 
sin, (ii) a small amount of profibrinolysin itself, i.e. of material 
which can be activated by streptokinase, and (iii) antifibrinolysiny 
an inhibitor of plasmin, which, however, is present in a much lower 
concentration in the seminal plasma than in blood serum. 

Fibrinogenase is the name given by Huggins and Neal (1942) 
to the proteolytic agent, highly active in canine prostatic secretion, 
but less so in human prostatic fluid, which destroys blood plasma 
fibrinogen. Huggins and his co-workers (1942, 1943) also share the 
credit for having recognized the similarity between certain other 
proteolytic properties of the prostatic secretion and those of pan- 
creatic trypsin. More recently, the 'tryptic' enzyme of human semen 
has been partially purified by Lundquist (1952), who defined as a 
unit the amount of enzyme which in 1 hr., at pH 7-6, and 37°, 
liberates from added casein a quantity of chromogen corresponding 
to 01 mg. free tyrosin; he achieved an activity of about 1 unit per 
mg. of protein-nitrogen, i.e. an approximately tenfold purification. 
The purified enzyme was active also towards haemoglobin and both 
human and bovine blood plasma fibrinogen. In an attempt to 
purify from human semen the natural substrate for the proteolytic 
activity, Lundquist obtained a protein fraction, 'seminal fibrin', 
which was readily digested by enzyme preparations both from 

1 1 6 The Biochemistry of Semen 

semen and the prostate gland, and yielded the same amino acids 
which appear normally in human semen. 


Apart from the above mentioned protease which acts optimally 
at pH 7-6, two more proteolytic enzymes have been found in 
human semen by Lundquist and his co-workers (1951, 1952, 1953). 
One is an amino peptidase which hydrolyses leucine amide, glycyl- 
glycine, triglycine and glycyl- leucine, with an optimum around pH 
7-5. The other has been identified as pepsinogen. According to 
Lundquist and Seedorff (1952), the activity of pepsinogen in semen 
corresponds to 2 //g. pepsin /ml., which is of the same order of 
magnitude as that found in gastric juice. But unlike the trypsin- 
like enzyme, seminal pepsinogen seems to originate in the seminal 
vesicles and not in the prostate. Its specific function is not fully 
understood as it is difficult to envisage in semen the high hydrogen 
ion concentration required for the conversion of pepsinogen to 

Ammonia formation 

A phenomenon probably associated with the enzymic degrada- 
tion of proteins, is the progressive accumulation of free ammonia 
which takes place in whole semen and in seminal plasma, on anaero- 
bic as well as aerobic incubation. This has been observed in several 
species (Shergin, 1933; Mann, 1945^). In ram semen for example, 
the content of free ammonia (estimated by vacuum-steam distilla- 
tion in the Parnas-Heller apparatus) was found to increase from 
1-3 mg. NH3-N/IOO ml. in fresh semen to 9-7 mg. NH3-N/IOO ml., 
after 7 hr. incubation at 37°, under sterile conditions (Mann, 
\9A5a). Ammonia formed in semen is in considerable excess of the 
amount which could be derived from adenyl derivatives; the total 
adenine amino-A'^ content of the semen as assessed enzymically 
with heart muscle deaminase being only about 4 mg./lOO ml. in 
fresh, and 3 mg./lOO ml. in incubated, semen. It is also unlikely to 
originate from urea, since the content of urea in semen is not sig- 
nificantly affected by incubation. 

Protein Constituents and Enzymes of Seminal Plasma 1 1 7 

Amino acid oxidase 

In addition to sugars and fatty acids, spermatozoa are capable 
of oxidizing a number of amines and amino acids. The oxidative 
deamination of amino acids by bull spermatozoa has been the subject 
of a study by Tosicand Walton (1945; 1946«, b, 1950). The starting 
point of this study has been the observation that the addition of 
egg-yolk to bull sperm causes an increased oxygen uptake, which, 
however, gradually declines in about an hour's time. Egg-yolk 
fractionation led to a dialysable, nitrogen-containing fraction which 
was oxidized by spermatozoa. The oxidation was accompanied by 
accumulation of ammonia and formation of hydrogen peroxide. 
Evidence was obtained which pointed to peroxide being responsible 
for the gradual decline in the oxygen uptake by spermatozoa. 

In the course of their study, Tosic and Walton examined several 
pure amino acids and found that spermatozoa oxidize three natur- 
ally occurring amino acids, namely L-tyrosine, L-phenylalanine and 
L-tryptophan. According to Tosic (1947, 1951), the hydrogen- 
peroxide-forming aerobic process in bull semen is an oxidative 
deamination catalysed by the L-amino acid oxidase of spermatozoa, 
which differs from the analogous enzyme of other animal tissues 
by having its range of activity restricted to only three aromatic 
amino acids; the activity of the enzyme can be expressed by the 


I "II 

NH2 O 

Seminal phosphatases 

Semen owes its powerful phosphatase activity mainly to the 
seminal plasma which carries several different dephosphorylating 
enzymes derived from the male accessory organs of reproduction. 
Among the most active and best known enzymes in this group are the 
so-called 'acid phosphatase' and 'alkaline phosphatase'. In addition 
to these two phosphomonoesterases, the seminal plasma contains 
'5-nucleotidase', a pyrophosphatase, and several adenosinetri- 

In early studies on phosphatases, the substrates commonly 

118 The Biochemistry of Semen 

used were a- and /3-phosphoglycerol, and phosphohexoses, chiefly 
6-phosphoglucose and 6-phosphofructose, but also 1 : 6-diphospho- 
fructose. More recently, however, other organic phosphoric acid 
derivatives came into use, including various nucleotides and inter- 
mediary phosphorylated compounds of glycolysis, as well as two 
synthetic substances: phenylphosphate (King and Armstrong, 1934) 
and phenolphthaleine phosphate (King, 1943; Huggins and Talalay, 
1945). The introduction of histochemical techniques marked another 
important development in studies on phosphatases (Gomori, 1939, 
\94la, b, 1953). The histochemical investigations have thrown much 
light upon the pattern of phosphatase distribution in the male 
accessory organs and have helped to establish the existence of 
'secretory' phosphatases, localized in the secretory epithelia and 
secretions of accessory glands, as distinct from the 'stromal' phos- 
phatases which are present only in the stroma (Dempsey, 1948; 
Bern, 1949; RoUinson, 1954). 

''Acid' and 'alkaline' phosphatase 

An observation that the phosphatase activity of male urine is 
usually higher than in women, led Kutscher and Wolbergs (1935) 
to examine the phosphatase in semen and in the prostate gland. 
They soon found that semen and prostate are among the richest 
sources of acid phosphatase in the human body, the enzyme being 
optimally active at pH 5-6, equally well towards a- and ^S-phospho- 
glycerol, but largely inactive towards diphosphofructose and pyro- 
phosphate (Kutscher and co-workers, 1936, 1938). Subsequent 
investigations confirmed and extended these findings; the demon- 
stration by Scott and Huggins (1942) that, while the voided urine 
of man is rich in the enzyme, urine collected directly from the renal 
pelvis shows only little enzymic activity, was a convincing proof 
that the content of acid phosphatase in normally voided male urine 
is due largely to the admixture of prostatic secretion. 

Acid phosphatase is an important secondary male sex charac- 
teristic. Investigations by Gutman and Gutman (1938Z)) have shown 
that the level of the enzyme in the human prostate is low in child- 
hood but increases rapidly at puberty; thus the activity, expressed in 
King- Armstrong units per gram prostate tissue, was H units at four 
years of age, 73 units at puberty, and 522 to 2284 units in adult men. 

Protein Constituents and Enzymes of Seminal Plasma 119 

A similar relation to age was observed in monkeys and dogs; in 
both these species administration of androgenic hormones to imma- 
ture males stimulates considerably the output of the enzyme from 
the prostate gland (Gutman and Gutman, 1939; Huggins and 
Russell, 1946). A certain correlation appears to exist in adult men 
between the level of acid phosphatase in semen and androgenic 
activity (Gutman and Gutman, 1940; Gutman, 1942; Engberg, 
Anderson, Sury and Raft, 1947). However, like other constituents 
of semen, the level of prostatic phosphatase activity varies from one 
species to another, as well as between individuals within the same 

Under physiological conditions, acid phosphatase does not pass 
from the prostate into the blood stream. However, significant 
amounts of it appear in the blood plasma as a result of malignant 
growth in the prostate and metastases of prostatic cancer in the 
bones; injections of androgen still further increase the level of 
enzyme in blood plasma, whereas castration or treatment with 
oestrogens lead to a spectacular decrease. The determination of 
prostatic phosphatase activity in blood has been utilized as a valuable 
diagnostic aid in prostatic carcinoma and in the course of clinical 
treatment (Gutman and Gutman, 1938a; Huggins, Scott and Hodges, 
1941; Watkinson, Delory, King and Haddow, 1944). 

An important addition to our knowledge of the physiological 
functions of acid phosphatase in seminal plasma, has been the dis- 
covery made by Lundquist (1946) that freshly ejaculated human 
semen contains phosphorylcholine which on ejaculation is rapidly 
dephosphorylated by the acid phosphatase to free choline and 
orthophosphate. This phenomenon is more fully discussed else- 
where (p. 170). But there is evidence that apart from the acid phos- 
phatase which acts on phosphorylcholine optimally at pH 6-3 (in 
acetate buffer), human seminal plasma contains another phosphatase 
which acts on the same substrate at a higher pH (Hudson and Butler, 
1950). Of considerable interest is also the finding that the acid 
seminal phosphatase exhibits in vitro a distinct transferase activity 
(Green and Meyerhof, 1952); partially purified acid phosphatase 
from the human prostate has been shown to catalyse at pH 5-5 
the transfer of phosphate from both ^S-phosphoglycerol and phospho- 
creatine to glucose; the product in each case was 6-phosphoglucose 


The Biochemistry of Semen 

(Morton, 1953). Methods for the purification of acid phosphatase 
from human prostate glands and semen have been described by 
London and Hudson (1953) and Boman (1954). 

Alkaline phosphatase, like the 'acid' enzyme, is widely distributed 
in male accessory organs but its localization in cells and concentra- 
tion in accessory gland secretions is different. Human semen with 
its conspicuously high level of acid phosphatase, has a low concen- 
tration of the alkaline phosphatase. Bull semen on the other hand, 
has only slight acid phosphatase activity but contains more of the 
alkaline phosphatase (Haq and Mullen, 1948; Reid, Ward and 
Salsbury, 1948^). This difference between the human and bovine 
semen is not altogether unexpected, since the bulk of bull seminal 
plasma is derived not from the prostate but from the seminal 
vesicles. In the rat both phosphatases are of low activity; with the 
exception of the ventral prostate which may contain up to 20 units 
of alkaline phosphatase per g. tissue, the level of either enzyme 
seldom exceeds 4 units per g. in any one of the other accessory 
organs. After castration the activity of both these enzymes diminishes 
first in the rat seminal vesicles, and a little later in the prostate; but 
the percentage decrease of enzymic activity and of organ weight is 
roughly equal (Huggins and Webster, 1948; Stafford, Rubinstein 
and Meyer, 1949). 

Table 17. Phosphatase activity of ram seminal plasma on 
phosphohexoses (Mann and Lutwak-Mann, 19516) 

(The liberation of sugars was examined by incubating 5 mg. substrate 
(Na salt) with 1 ml. dialysed seminal plasma at pH 7 or 0-2 ml. dialysed 
seminal plasma at pH 9, for 1 hr., 37°, in the presence of OOOSM-MgCla. 
The sugars were determined after deproteinization with ZnS04 and 
Ba(OH)2; glucose was estimated by means of glucose oxidase (Mann, 
1944; \9A6b).) 

Fructose (%) 

Glucose (%) 

Inorg. phosphate (%) 





pH=7 pH=9 






67 93 






94 100 






97 100 






94 100 

1 : 6-Diphosphofructose 





20 95 





60 100 

Protein Constituents and Enzymes of Seminal Plasma 121 

Alkaline phosphatase has an optimum at about pH 9, and is 
capable of hydrolysing among others, 1-phosphofructose, 6-phos- 
phofructose and 1 : 6-diphosphofructose. Our researches indicate 
that this activity may represent an essential step in the process of 
fructose formation and secretion by the accessory organs (Mann 
and Lutwak-Mann, 1951«, b) (see also p. 150). Ram seminal plasma 
is particularly rich in alkaline phosphatase which, though it acts 
optimally at pH 9, also shows appreciable activity towards phos- 
phohexoses at pH 7. This can be seen from Table 17 which gives 
rates of dephosphorylation for various compounds. In the case of 
6-phosphofructose and 1 : 6-diphosphofructose, some glucose is 
formed in addition to fructose, owing to the presence of phospho- 
hexose isomerase in the seminal plasma, as a result of which a part 
of 6-phosphofructose is converted to 6-phosphoglucose before 
dephosphorylation. Supporting evidence for the conclusion that 
monophosphofructose rather than diphosphofructose, is the sub- 
strate immediately responsible for the liberation of seminal fructose, 
has been provided by Bouchilloux and Menager (1952) who found 
that the semen of both ram and bull contains two phosphomono- 
esterases, with pH optima at 9-4 and 4-8, respectively, but that it 
lacks a specific fructosediphosphatase. 


This enzyme was discovered in human semen by Reis (1937, 
1938, 1940) and shown to dephosphorylate muscle adenylic acid 
(adenosine-5 '-phosphoric acid) and inosinic acid (inosine-5'-phos- 
phoric acid) but not adenosine-3 '-phosphoric acid or adenosine 
triphosphoric acid. Bull seminal plasma is particularly rich in 5- 
nucleotidase, the seminal vesicles being the main source of the 
enzyme (Mann, 1945a, 1947). Bull seminal plasma or the vesicular 
secretion itself, act several hundred times more efficiently on muscle 
adenylic acid than on /S-phosphoglycerol; from 160 ^g. P added as 
sodium adenylate to 0001 ml. bull seminal plasma, up to 140 /^g. 
P are liberated as orthophosphate during 1 hour's incubation at 37°. 

The 5-nucleotidase of bull seminal plasma has been purified about 
fifty-fold by Heppel and Hilmoe (195 la). The purified enzyme 
has a pH optimum at 8-5, and its activity is enhanced by the addi- 
tion of magnesium ions but inhibited by fluoride (00 1m) and by 

122 The Biochemistry of Semen 

borate buffer (0-08m) to the extent of 73 and 85% respectively. On 
the basis of tests with numerous phosphorylated compounds, it 
may be safely concluded that 5-nucleotidase is an enzyme which acts 
specifically on substrates containing the ribose-5-phosphate moiety. 
The purified enzyme splits rapidly ribose-5-phosphate but not 
ribose-3-phosphate; it is active towards adenosine-5 '-phosphate, 
inosine- 5 '-phosphate, uridine-5'-phosphate and cytidine-5'-phos- 
phate but inactive towards both adenosine-3 '-phosphate and 
adenosine-2'-phosphate. It also dephosphorylates nicotinamide 
mononucleotide (nicotinamide ribose-5'-phosphate); this inciden- 
tally explains an early observation of ours, that bull seminal plasma 
decomposes cozymase, with slow liberation of inorganic phosphate. 


Bull seminal plasma contains an enzyme which hydrolyses in- 
organic pyrophosphate to orthophosphate but differs from the 
pyrophosphatase of yeast. Seminal pyrophosphatase can exert its 
maximal activity in the absence of magnesium ions and is not 
inhibited by increased substrate concentration; it has a sharp 
optimum at pH 8-6 (Heppel and Hilmoe, \95\b), 

Enzymic hydrolysis of adenosine triphosphate 

In addition to adenosine-triphosphatase (ATP-ase) in the sperma- 
tozoa there are also ATP-splitting enzymes in the seminal plasma 
(Mann, 1945o; MacLeod and Summerson, 1946). When adenosine 
triphosphate is acted upon by bull or human seminal plasma, all 
three phosphate groups are set free as orthophosphate. The mechan- 
ism of this reaction has been investigated by Heppel and Hilmoe 
(1953) who by fractionation procedures obtained three distinct 
ATP-ases, none of them, however, completely free from 5-nucleo- 

One of the enzymes, named the 'pyrophosphate-forming ATP- 
ase', catalyses the reaction 

Adenosine triphosphate +H2O — >- 


It is relatively heat-stable, has a pH optimum at 8 -4-8 -6 and 
requires neither calcium nor magnesium ions for activation. The 

Protein Constituents and Enzymes of Seminal Plasma 123 

remaining two ATP-ases, designated respectively as 'acid' and 
'alkaline', produce orthophosphate; the 'acid' ATP-ase has a pH 
optimum at 5-7-60, requires magnesium, is inhibited by calcium, 
and can be inactivated completely by heating for 20 min. at 60°; it 
does not act on i5-phosphoglycerol; the 'alkaline' ATP-ase is more 
heat-resistant, has a pH optimum at 8 -4-8 -8, and is stimulated by 
calcium and also by magnesium. Both these enzymes are active not 
only tov^ards adenosine triphosphate but adenosine diphosphate 
as v^ell. 

Bull seminal plasma contains in 1 ml. about 80 units of acid 
ATP-ase, 130 units of alkaline ATP-ase, 40 units of the pyrophos- 
phate-forming ATP-ase, and 2900 units of 5-nucleotidase. It re- 
mains for future studies to define the physiological significance 
of all these enzymes, particularly the 5-nucleotidase which is so 
characteristic of semen. Possibly, there is some link between them 
and other nucleolytic enzymes and they may well play a role in the 
metabolism of purine compounds in semen and reproductive organs. 
The occurrence of nucleases in human and sea-urchin semen 
(Zamenhof, Shettles and Chargaff, 1950; Mazia, 1949), the cozy- 
mase-destroying activity of bull seminal plasma (Mann, 1945a), the 
interesting findings on the presence of uric acid in bull semen 
(Barron and Haq, 1948; Leone, 1952), and the more recent demon- 
stration of xanthine oxidase in the bull vesicular secretion (Leone, 
1953), are but a few examples of problems in this field, which await 
further and more detailed study. 


Lipids and their Role in the 
Metabolism of Semen 

Lipids in spermatozoa. The lipid capsule. Acetal phospholipids or plas- 
malogens. Role of lipids in sperm metabolism. Lipids in the seminal plasma 
and male accessory gland secretions. 'Lipid bodies' and prostatic calculi. 

Lipids in spermatozoa 

The first systematic analysis of lipids in spermatozoa was carried 
out by Miescher (1878, 1897) who also proved that the lipids are 
concentrated chiefly in the sperm-tails. His analytical results showed 
that the ether-extractable material obtained from salmon sperma- 
tozoa is composed of about 50% lecithin, 14% cholesterol and 
35% fat, and that by far the greatest part of this material is derived 
from the sperm-tails where lecithin accounts for 31-83%, fats and 
cholesterol for 26-27%, and protein for the remaining 41 -90% of the 
organic contents. This led Miescher to conclude that the sperm- 
tails resemble in their composition the grey matter of the nervous 
system, and in a letter to W. Hiss he wrote: 'The more I deal with 
the tails, the more probable it appears to me that we have before 
us essentially the chemical type of the non-medullated nerves, that 
is the axis cylmders.' Subsequent investigations by Mathews (1897) 
and Sano (1922) on the sperm of herring, salmon, porgy and cod- 
fish, confirmed the presence of lecithin and revealed at the same 
time the presence of small quantities of certain other lipids, includ- 
ing cephalin and sphingomyelin. 

H2C — O — CORunsat. 

HC— O— CORsat. Lecithin 


H2C— O— P— O— CH2-CH2 

I I 

O- +N(CH3)3 


Lipids and their Role in the Metabolism of Semen 125 

When air-dried salmon roe is ground and extracted with pentane 
in a Soxhlet apparatus, a yellow oil is obtained which is practically 
free from phospholipids; it requires further treatment with ethanol 
or methanol for the phospholipids to be extracted from the sperm. 
In this way, for example, 12-5% glyceride in the oily fraction, and 
6-2% phospholipid in the alcoholic fraction, was obtained from the 
roe of the sockeye salmon, Oncorhynchus nerka (Halpern, 1945). On 
the basis of this observation, it has been suggested that the phos- 
pholipids occur in the spermatozoa in a firmly bound state, pre- 
sumably in the form of lipoproteins. A substantial portion of the 
unsaponifiable material extracted from fish sperm by fat solvents 
consists of cholesterol which accounts, on the average, for 2-2% 
of dried fish spermatozoa (Schmidt-Nielsen and Sundsvold, 1943). 

The high content of lipids in spermatozoa is equally characteristic 
for fishes as for other animals. Sea-urchin spermatozoa are well 
known to contain a large reserve of lipid material, shown by Mathews 
(1897) to include both lecithin and neutral fat. The content of phos- 
pholipids in the sperm of Echinus esculent us is about 5-5% of the 
dry weight of spermatozoa (Rothschild and Cleland, 1952). The 
seminal lipids of E. esculentus have been analysed more recently by 
Cardin and Meara (1953). The material obtained by extraction of 
1-2 1. of semen with acetone and light petroleum, consisted of 13-6% 
neutral fat, 32-9% free fatty acids, 260% phospholipids, 9-2% 
sterols and 18-3% of other unsaponifiable matter. The component 
fatty acids of the non-phospholipid fraction included a low propor- 
tion (10-1%) of saturated acids and a high proportion of unsatur- 
ated acids with 18C (30-4%) 20C (451%) and 22C (12-3%). The 
phospholipid fraction had a ratio of N : P=l-4 : 1, and must have 
therefore, consisted of a mixture of monoaminophosphatides and 
diaminophosphatides . 

Early analyses of lipids in bovine epididymal sperm were carried 
out by Koelliker (1856) who found that over 12% of the dried 
material is ether-extractable. About half of this content was later 
shown by Miescher (1878) to consist of lecithin. In a study of the 
lipid content of bull sperm, Zittle and O'Dell (1941«) have extracted 
washed epididymal spermatozoa successively with ethanol, ether, 
acetone and petroleum ether, and found 13% of lipid material; when 
the procedure was repeated with spermatozoa disintegrated by sonic 

126 The Biochemistry of Semen 

treatment, more lipid was found in the tails (23%) than in the mid- 
pieces (6%) or heads (7%). 

The lipid capsule 

It is probable that the high lipid content of spermatozoa is due 
largely to the lipid 'sheath' or 'capsule' which encloses the sperm 
cell. So it would seem at any rate, from histochemical studies, 
including the extensive investigation of Popa and Marza (1931) who 
described the so-called manteau Upidique in the spermatozoon of 
man, dog, bull, ram, boar, rabbit, guinea-pig and cock. The lipid 
capsule is presumably of importance to the spermatozoa in their 
function, perhaps to ward off the effects of the acid vaginal milieu as 
has been suggested by Redenz (1924). It appears to consist largely 
of a lipoprotein complex which is fairly soluble in aqueous solvents. 
In the case of mammalian spermatozoa, this complex has been 
extracted with a 01 4m solution of sodium chloride at pH 9 (Dallam 
and Thomas, 1952). In the middle-piece lipids were shown to be 
associated with the 'spiral body' which surrounds the axial proto- 
plasmic thread, and is derived from the mitochondria of the sperma- 
tids (Wislocki, 1950; Brown, 1952). 

Acetal phospholipids or plasmalogens 

An interesting feature of the sperm cell is a characteristically high 
content of acetal phospholipids or plasmalogens. Feulgen and 
Rosenbeck (1924) while studying the 'nucleal' reaction of human 
spermatozoa, noted that when fresh smears of human semen were 
treated with Schiff's fuchsin-sulphurous acid reagent, the middle- 
pieces and tails, though devoid ojf nuclear material, stained strongly. 
This observation was followed by a demonstration that cells in 
general contain in their protoplasm some material which stains 
diffusely with Schiff's reagent, but differs from nucleoproteins by its 
solubility in ethanol. The name 'plasmal' was bestowed upon this 
material, which was shown in subsequent investigations by Feulgen 
and his co-workers to arise from 'plasmalogen', a group of peculiar 
phospholipids widely distributed in tissues, and distinguished by the 
presence of higher fatty aldehydes in place of the usual fatty acids. 
The plasmalogen isolated by Feulgen and Bersin (1939) from beef 
muscle was identified as an acetal of glycerylphosphorylcolamine. 

Lipids and their Role in the Metabolism of Semen 127 

The two principal fatty aldehydes in plasmalogens are palmitic and 
stearic aldehydes but other fatty aldehydes were also reported 
(Feulgen, Boguth and Andresen, 1951). Crystalline acetal phos- 
pholipids were prepared from beef brain, and shown to belong to 
the a-series (Thannhauser, Boncoddo and Schmidt, 1951). 

H.C— O 


HaC — O Acetal a-phospholipid 

I O 


H,C— O— P— O— CH, 

I 1 


The plasmalogen content of bull semen as determined by Boguth 
(1952) was found to vary from 30 to 90 mg./lOO ml.; of this about 
two-thirds is present in the sperm and one-third in the seminal 
plasma. The volume taken up by sperm in bull semen is compara- 
tively small, about 10%; it would seem therefore, that the concentra- 
tion of the acetal phospholipids in the spermatozoa themselves must 
be of the order of 200-600 mg./lOO g. fresh weight, or 3 x lO-^" mg. 
per cell. 


The list of interesting chemical substances which occur in semen 
was extended again when in 1941 Wagner- Jauregg reported on the 
isolation of the hydrocarbon heptacosane from human semen. An 
alcoholic extract obtained from 18 litters of semen formed, upon 
concentration in vacuo, a solid residue which was extracted first 
with 1-5 1. acetone, and next with 2 1. of a mixture of equal amounts 
of ethanol and ether. On purification, the acetone-soluble fraction 
yielded some crystalline material which melted at 57-60° and con- 
sisted, in all probability, of palmitic and stearic acid. The ethanol- 
ether soluble fraction formed on standing a crystalline precipitate 
containing 1 g. of heptacosane, CH3(CH2)25CH3, which on recrystal- 
lization showed the required melting point, 59-5°. The isolation of 
heptacosane has previously been achieved from plant material. It is 
also known to be associated in a characteristic manner with beeswax. 

128 The Biochemistry of Semen 

So far, however, the only instance other than semen, where hepta- 
cosane has been shown to occur in the human body, is in the urine 
of pregnant women. It is absent from the urine of men and of non- 
pregnant women, and is devoid of oestrogenic activity. Nothing 
is known about the origin or function of seminal heptacosane. 
Should future investigations, however, show that heptacosane in 
semen is involved in the metabolism of fatty acids or aldehydes, then 
its fate would be analogous to that of plant hydrocarbons which 
are well known to be associated with the metabolism of fatty acids, 
aldehydes and alcohols in plants. 

Role of lipids in sperm jnetabolism 

The functional aspects of lipid metabolism in spermatozoa have 
been the subject of investigations by Lardy and Phillips (1941«, b\ 
1945). To begin with, these authors confirmed the observation 
originally made by Redenz (1933) that, in contrast to whole semen 
which can be stored successfully both anaerobically and aerobically 
owing to the presence of glycolysable carbohydrate in the seminal 
plasma, bull spermatozoa separated from the seminal plasma by 
centrifugation and washing, can survive only in the presence of 
oxygen. From this they inferred that when the spermatozoa are 
deprived of sugar, they begin to oxidize aerobically some of their own 
intracellular constituents as a source of energy for motility. To 
detect the oxidizable substrate, sperm samples were analysed when 
fresh and after periods of storage; it was then found that a period 
of aerobic incubation of bull spermatozoa caused a significant de- 
crease in the content of lipid phosphorus accompanied by an increase 
in the acid-soluble phosphorus. But when glucose was added to the 
washed sperm, the decrease in the phospholipid content of sperma- 
tozoa was very slight, an indication perhaps, of a preference by 
spermatozoa for the glycolytic mechanism as a source of energy 
(Table 18). It was also found that certain phospholipids prepared 
from egg-yolk, liver and soya bean, effectively maintained the 
oxygen uptake and motility of washed sperm suspensions under 
aerobic conditions; however, on the addition of sugar to the sperm 
suspension, the phospholipids no longer produced an effect on 
either respiration or glycolysis. 

In bull semen as ejaculated, with its large reserve of readily 

Lipids and their Role in the Metabolism of Semen 129 

Table 18. Changes in the phospholipid content of bull spermatozoa 
in presence and absence of sugar (Lardy & Phillips, \9A\a) 

(Bull spermatozoa freed from seminal plasma by centrifugation, then 
diluted with Ringer-phosphate solution to the original volume of semen, 
and incubated at room temperature.) 

Medium Phospholipid content 

After 10 hr. 

Original incubation 

(mg. P/ml.) (mg. P/ml.) 

Ringer-phosphate 0-38 0-24 

Ringer-phosphate+0-04M-glucose 0-39 0-37 

glycolysable material in the form of fructose, the share of phos- 
pholipids in sperm metabolism is probably small. But in the epi- 
didymis, where glycolysable sugar is unavailable, Lardy and Phillips 
ascribe great importance to the phospholipids as a source of oxida- 
tive energy. The mechanism of utilization of this reserve is held to 
involve hydrolytic cleavage of phospholipids foUov^ed by an oxida- 
tion of the fatty acid portion via the citric acid cycle, and coupled 
with aerobic phosphorylations. According to Lardy, Hansen and 
Phillips (1945), the aerobic metabolism of phospholipids in the 
bovine epididymal spermatozoa is accompanied by an uptake of 
inorganic phosphate, and the formation of a phosphate ester which 
is hydrolysed in 7 min. by N-HCl at 100°, and thus resembles 
adenosine triphosphate. 

The ability to utilize phospholipids as a source of aerobic energy 
extends to the spermatozoa of lower animals, notably those of the 
sea-urchin. One of the main differences between mammalian and 
sea-urchin semen is that the latter contains no glycolysable material 
in the seminal plasma. The possibility that sea-urchin spermatozoa 
which have been shed into sea- water, survive at the expense of 
energy derived from the oxidation of intracellular carbohydrate such 
as glycogen, also appears remote, in view of the very low content 
of glycogen-like material in the sperm cells (Stott, 1930; Rothschild 
and Mann, 1950). On the other hand, according to Rothschild and 
Cleland (1952), the content of intracellular phospholipids which in 
fresh sperm of Echinus esculentus is 5-5% of the dry weight or 4-14 
mg. (0165 mg. P) per 10^*^ sperm cells decreases in the course of 
aerobic incubation of sperm suspensions in sea-water, at an average 

130 The Biochemistry of Semen 

rate of 0-787 mg./lO^" sperm cells/7 hr. The oxygen uptake recorded 
during the same period is 1-45 ml. Oa/lO^" sperm, which if sustained 
exclusively by phospholipids, would require the disappearance of 
0-906 mg. of phospholipid. On the basis of these observations 
Rothschild and Cleland conclude that the principal source of energy 
required for the movement of sea-urchin spermatozoa is derived 
from the oxidative breakdown of phosphilipids located mainly in 
the middle-piece of the sperm cell. 

Lipids in the seminal plasma and male accessory gland secretions 

Apart from the lipids which form a part of the sperm structure, 
there is also some lipid material in the seminal plasma. The bulk of 
the 'bound choline', however, does not consist of phospholipids 
but occurs in the form of acid-soluble phosphorylated derivatives of 
choline (see p. 170). The lipid of the human seminal plasma origin- 

Table 19. Lipids of the human prostatic fluid and seminal plasma 
(w^./lOO ml.) (Scott, 1945) 

(No. indicates the number of studied specimens.) 

Prostatic fluid 

Seminal plasma 



Lipid fraction No. 








Total lipid 10 








Total phosphatide 10 








Moist ether-soluble 

phosphatides 10 








Lecithin 10 


Cephalin 10 








Moist ether-insoluble 

phosphatides 10 








Total cholesterol 10 








ates chiefly from the prostatic fluid. Moore, Miller and McLellan 
(1941) analysed twelve specimens of human prostatic secretion and 
found up to 9-5 mg. lipid phosphorus per 100 g. fluid, with an 
average of 2-7 mg. P/100 g. or 67-5 mg. phospholipid/ 100 g. Scott 
(1945), whose analytical results are shown in Table 19, found an 
average content of 286 mg./lOO ml. of 'total lipid' and 179-8 
mg./lOO ml. of phospholipid, in the human prostatic secretion; 

Lipids and their Role in the Metabolism of Semen 131 

and 185-5 mg./lOO ml. of 'total Upid' and 83-5 mg./lOO ml. of 
phospholipid, in the seminal plasma. However, he was unable to 
detect lecithin either in the prostatic fluid or in the seminal plasma. 
In both instances, two-thirds of the phospholipid consisted of an 
ether-soluble choline-free phosphatide, probably identical with cepha- 
line, the rest being some other, ether-insoluble material. Scott 
found little neutral fat in either the prostatic secretion or the seminal 
plasma, the sum of phospholipids and cholesterol accounting for 
practically the entire 'total lipid'. The content of 70-120 mg. total 
cholesterol /1 00 ml. seminal plasma recorded by Scott, is below the 
cholesterol value for human blood plasma; a similar figure, 80 mg./ 
100 ml., has been reported earlier by Goldblatt (1935a). 

'Lipid bodies'' and prostatic calculi 

In many species, the seminal plasma contains small globules, 
droplets or granules, sometimes called the 'lipid bodies'. In man, 
dog, cat, and rabbit, they are derived chiefly from the prostatic 
secretion but in certain species they occur also in the seminal vesicle 
secretion (Prevost and Dumas, 1824; Pittard, 1852). The globules of 
the human prostatic secretion are referred to by Sir Henry Thompson 
in his famous prize essay on the Diseases of the Prostate (1861), 
as 'small yellowish bodies, in appearance sometimes granular, some- 
times homogeneous, about the size of red blood corpuscles, but not 
so uniform, being from about 1/5000 to 1/2500 of an inch in 
diameter' and exhibiting 'considerable refractive power nearly so 
much as to give them a resemblance to oil globules'. The occurrence 
of similar elements in the prostatic secretion was later observed by 
Fuerbringer (1881, 1886) who coined for them the name Leclthin- 
kdrnchen\ These 'lecithin granules' or 'lecithin bodies' which Fuer- 
bringer regarded as responsible for the normal opalescence and 
milky appearance of the prostatic fluid, have since been re-examined 
on several occasions, mostly by means of histological methods. 
Chemical analysis however, failed to corroborate the presence of 
lecithin in these particles. Other curious structures which according 
to some authors are closely linked with the appearance of 'lipid 
bodies' in the human seminal plasma, are certain larger bodies 
known as 'colostrum corpuscles', 'corpora amylacea' and 'pros- 
tatic calculi'. The colostrum corpuscles, frequently met with in the 

1 32 The Biochemistry of Semen 

human prostatic secretion, are macrophages packed with masses of 
lipid granules which stain strongly red with eosin. The corpora 
amylacea are small, soft, concentrically laminated spheroidal bodies, 
pale yellow to dark brown in colour, frequently, though not invari- 
ably, doubly refractile. They are usually located in the larger ducts 
and acini of the prostate and are probably made up of desquamated 
epithelial cells and prostatic secretion. They have been shown to 
contain some cholesterol but according to Moore and Hanzel 
(1936) the double refraction may be due to certain purines, decom- 
position products of nucleoproteins, and not to lipids. The prostatic 
calculi are ordinarily not more than a few mm. in diameter but 
occasionally they may replace the whole prostatic parenchyma. 
They are firm, calcified bodies, the basic structure of which, except 
for size and infiltration by calcium salts, is apparently the same as 
that of the corpora amylacea (Moore, 1936). Wollaston (1797) 
described them as composed of 'phosphorated lime in the state of 
neutralization, tinged with the secretion of the prostate gland'. He 
was also the first to show that they are not urinary products. In 
recent times, the chemical composition of prostatic calculi has been 
investigated by Huggins and Bear (1944); a considerable proportion 
of the prostatic stones was inorganic and consisted of calcium and 
magnesium phosphates and carbonates but there was also some 21 % 
of organic matter composed of protein, citrate and cholesterol 
(Table 20). 

Although characteristic of the human prostate, corpora amylacea 
are also found elsewhere, particularly among insectivores where 
their production is considered to be one of the chief secretory 

Table 20. Chemical analysis of prostatic calculi (Huggins, 1947) 

(Stones from 6 men; average values expressed in % of dry powdered 








Phosphorus as PO4 


Carbon dioxide 


, Protein (Nx 6-25) 


Citric acid 




Lipids and their Role in the Metabolism of Semen 133 

functions of the prostate gland (Hopkins, 1911; Eadie, 1948^, b). 
They do not seem to occur either in the dog or the rat. The total 
lipid content of the dog prostatic fluid ranges from 30 to 40 
mg./lOO ml. (Huggins, 1947) and the lipid phosphorus from M 
to 2-2 mg. P/100 ml. (Moore et al, 1941). There exists a condition 
known as the 'benign prostatic hypertrophy', which is common to 
dog and man. In the dog, however, this condition is not associated 
with the occurrence of corpora amylacea or any other spheroidal 
nodules but consists of cystic hyperplasia (Huggins and Clark, 
1940). In the bull, lipid-laden cells form a highly characteristic 
component of the seminal vesicle epithelium, and the cavities of 
the tubules in the seminal vesicles contain an abundance of eosino- 
philic granular secretion (Mann, Davies and Humphrey, 1949). The 
extent to which organs other than the seminal vesicle and prostate, 
contribute to the lipid or sterol content of semen, has not been 
hitherto studied in much detail. In this connection, however, an 
interesting observation of Ward and Moore (1953) deserves to be 
mentioned, concerning the occurrence of 7-dehydrocholesterol in 
the preputial gland and epididymis of rat. 


Fructose and Fructolysis 

Fructose as a normal constituent of semen. Species differences. Site of 
formation. Seminal fructose as an indicator of male sex hormone activity; 
the 'fructose test' and its application to certain problems of sex endocrin- 
ology. Role of hypophysis. The relationship between blood glucose and 
seminal fructose. Effect of malnutrition. The enzymic mechanism of fruc- 
tose formation. Anaerobic and aerobic utilization of carbohydrate by 
spermatozoa. Pasteur effect and the 'metabolic regulator'. Intermediary 
reactions in sperm fructolysis and the role of phosphorus-containing 

There has been little precise knowledge about fructose (laevulose) 
in man and higher animals except the evidence of its occurrence in 
certain embryonic fluids and in metabolic dysfunctions like fruc- 
tosuria. The presence of a laevorotatory constitutent in foetal fluids 
was first noted by Claude Bernard (1855) but its chemical identity 
was not recognized until some time later when it was shown that 
fructose was a normal constituent of allantoic and amniotic fluid, 
foetal blood and the urine of new-born animals (Majewski, 1858; 
Griiber and Griinbaum, 1904; Paton, Watson and Kerr, 1907; 
Langstein and Neuberg, 1907; Orr, 1924; Cole and Hitchcock, 
1946; Bacon and Bell, 1948; Hitchcock, 1949). More recently, the 
source of foetal fructose was traced to the placenta (Huggett, 
Warren and Warren, 1951). So far as adult man is concerned, it 
was believed that in general the occurrence of fructose is restricted 
to pathological conditions; fructose has been demonstrated in 
transsudates, and in the urine of diabetics and persons suffering 
from the peculiar metabolic disorder known as 'spontaneous fruc- 
tosuria', the aetiology of which remains obscure. In the normal 
human or animal organism, fructose has been found to be utilized 
chiefly after enzymic conversion to glucose and glycogen; liver, 
kidney and the gastro-intestinal tract were shown to be the main 
sites of this process (Oppel, 1930; Bollman and Mann, 1931; 
Stewart and Thompson, 1941; Deuel, 1936; Reinecke, 1944). 


Fructose and Fructolysis 135 

Thus, in the light of the evidence available until relatively recently, 
it seemed rather improbable that in the normal, fully-developed 
mammalian organism, fructose could occupy a place on the list 
of 'animal carbohydrates', or that any specific function could be 
assigned to this sugar. 

Fructose as a normal constituent of semen 

Since the early researches on mammalian semen by McCarthy, 
Stepita, Johnston and Killian (1928), Ivanov (1931), Huggins and 
Johnson (1933) and other pioneers in the field of semen biochemistry, 
it was known that in several species, including man, a reducing and 
yeast-fermentable sugar is normally present in semen, the concen- 
tration of this sugar exceeding by far that of glucose in blood. 
However, up to 1945, in the extensive literature dealing with the 
subject of seminal sugar, this substance has been described either as 
glucose or simply as the reducing sugar of semen (Killian, 1933; 
Bernstein, 1933; Goldblatt, 1935«; Shergin, 1937; McKenzie, Miller 
and Bauguess, 1938; Davis and Cole, 1939; Moore and Mayer, 
1941; MacLeod and Hotchkiss, 1942; Salisbury and VanDemark, 
1945), and the only reference to a probable occurrence of fructose 
in semen is found in an early paper by Yamada (1933) who in a 
general survey of human tissues and body fluids carried out numer- 
ous fructose determinations by means of a colour reaction with the 
drug 'cryogenine'; of course, like so many colour tests, this reaction 
by itself cannot be regarded as specific for fructose, since it gives 
a positive result not only with fructose but also with other ketoses, 
nor does it distinguish between free fructose, that is D(-)-fructo- 
pyranose (formula in Fig. 14), and bound fructose, i.e. fructofura- 
nose, such as occurs for example, in the various phosphofructoses . 

In 1945, in the course of studies on the metabolism of semen, the 
seminal sugar was isolated for the first time and identified by chemi- 
cal methods as free D(-)-fructose (Mann, \9A5b\ \946a, b, c). The 
actual final isolation was accomplished with a 120 ml. sample of bull 
semen representing some thirty pooled bull ejaculates, which were 
collected within a twelve-hour period by the various Centres for 
Artificial Insemination of Cattle in England, and immediately des- 
patched to Cambridge. The chemical procedure involved the fol- 
lowing steps: (a) the preparation of methylphenyl-fructosazone, a 

136 The Biochemistry of Semen 

crystalline compound which has been shown by Neuberg (Neuberg, 
1902, 1904; Neuberg and Strauss, 1902; Langstein and Neuberg, 
1907; Neuberg and Mandl, 1946) to be one of the few chemical 
derivatives by means of which fructose can be identified and dis- 
tinguished from glucose and from other closely related sugars; {b) the 
purification of seminal fructose up to the point when it reached the 
specific optical activity of pure crystalline fructose: [aP°°= -92-2°; 
(c) the demonstration that fructose occurs in the semen in free form 
and that it accounts for the whole of the yeast-fermentable carbo- 
hydrate which yields 'ketose reactions' with resorcinol (Seliwanoff, 
1887; Roe, 1934), diphenylamine (Ihl, 1885) and similar colour- 
producing substances (Pinoff, 1905; Thomas and Maftei, 1927; 
Pryde, 1946); {d) proof obtained with the highly specific enzyme, 
glucose oxidase, that in semen glucose is either absent or present in 
mere traces. 

On the basis of the above findings, which excluded the presence 
of glucose, bound fructose, and other ketoses, a rapid colorimetric 
method has been developed by means of which it is possible to 
determine accurately the fructose content of semen; 05-0 1 ml. 
suffices for analysis of human, bull, ram or rabbit semen (Mann, 
1948a, b\ 1952). 

Species differences 

The following mammalian species have been found to contain 
fructose in semen: man, bull, ram, boar, stallion, goat, opossum, 
rabbit, guinea-pig, rat, mouse, hamster (Mann, 1949). Among the 
lower animals, fructose was found in the semen of an elasmobranch 
(the dogfish, Scylliorhinus caniculus) and in the reproductive organs 
of the male (but not female) grasshopper, Locusta migratoria 
(Humphrey and Mann, 1948; Humphrey, 1949). In this connection 
it is worthwhile to recall the occurrence of fructose in the haemo- 
lymph of the larvae of another insect, Gastrophiliis intestinalis 
(Levenbook, 1947). 

There are, however, considerable quantitative differences between 
the various species. In the bull and goat, for example, the con- 
centration of fructose in semen sometimes reaches a level of 
1000 mg./lOO ml., but in the boar and stallion it seldom exceeds 
50 mg./lOO ml. Human semen occupies an intermediate position as 

Fructose and Fructolysis 137 

can be seen from Tables 4 and 5, which include values for fructose 
in several species. But, when comparisons are made between a species 
with fructose-rich semen (bull) and one notoriously poor in seminal 
fructose (boar), it must not be forgotten that the volume of a single 
boar ejaculate is almost a hundred times that of a bull, so that in 
effect, a single ejaculate of either species contains about the same 
absolute amount of fructose. There are species, however, in which 
fructose is altogether absent from semen or present only in traces, 
and it is through the study of these animals that we may hope to 
gain insight into the problem of alternative sugars in semen. Cock 
semen for example, has no fructose or a negligible amount only, but 
it contains a certain amount (20-100 mg./lOO ml.) of anthrone- 
reactive material of which a variable fraction disappears on 
oxidation with glucose oxidase and must therefore, be identical 
with glucose (Mann and Hancock, 1952). Rabbit semen, unlike 
that of bull, ram and man, contains occasionally an appreciable 
admixture of glucose in addition to fructose (Mann and Parsons, 

Site of formation 

The reason for the conspicuous species differences in the concen- 
tration of fructose as well as the individual fluctuations (Table 4 
and 5), is the fact that fructose is a product not of the testes, but 
of the male accessory organs of reproduction, principally the seminal 
vesicles (Mann, \9A6b). Naturally, the highly variable anatomical 
characteristics of these glands such as their size, actual storage 
capacity, and secretory ability, are important factors which deter- 
mine the final output of fructose in the ejaculate (Fig. 4). All these 
considerations are pertinent to studies of human semen because of 
the exceptionally large individual variations in the secretory func- 
tion of the seminal vesicles and their rather small storage capacity 
which explains why the collection of consecutive ejaculates within 
a few days, usually yields samples with a conspicuously low level 
of fructose. It appears that a time interval of about two days is 
required to replenish the store of fructose in the vesicular secretion of 
man. Unlike in certain other mammals, the human seminal vesicle 
and vas deferens open into the urethra through a common channel 
known as the ejaculatory duct. Consequently, any obstruction at the 

138 The Biochemistry of Semen 

level of the ejaculatory ducts will prevent both fructose and sperma- 
tozoa from reaching the urethral canal. This fact has been aptly 
chosen as an aid to medical diagnosis by Young (1948, 1949) 
who described the case of a man in whom repeated semen analysis 
failed to detect fructose or sperm, although testicular biopsy re- 
vealed normal spermatogenesis; the case has been diagnosed as 
congenital bilateral aplasia of the vasa deferentia. 

It must be also mentioned that though the seminal vesicles are 
the main source of fructose in the higher mammals, yet an addi- 
tional small amount of this seminal sugar is derived from the 
ampullar glands (Mann, 1948^), and in some animals also from 
certain other glands. Thus, in the rabbit, fructose was located both 
in the glandula vesicularis (a structure corresponding to seminal 
vesicles) and in the ampullae, as well as in the prostate (Davies and 
Mann, \9Alb). The rat provides an instance of particular interest, 
as in this rodent the seminal vesicles are free from fructose alto- 
gether; instead, fructose is found in the dorso-lateral prostate and 
in the so-called coagulating gland, a small organ immediately 
adjacent to the seminal vesicles proper, with which it shares a 
common peritoneal sheath (Humphrey and Mann, 1948, 1949). 

Since fructose is produced by the accessory glands, and not the 
testes, it is not surprising that in whole fresh semen there is no 
direct proportion between fructose concentration and sperm density. 
On the contrary, both in man and in domestic animals, an inverse 
ratio between fructose and sperm concentration in semen is fre- 
quently met with; the simplest interpretation is that in a dense 
sample of semen the space occupied by the sperm cells is relatively 
larger, and the volume taken up by the fluid portion, i.e. the 
fructose-containing seminal plasma, correspondingly less. This 
factor has a direct bearing on the interpretation of laboratory 
examinations concerned with semen and male fertility or sterility. 
It explains, for instance, why a semen sample with a high content 
of fructose need not necessarily be one of good sperm quality, and 
furthermore, why it is possible to come across samples with a high 
fructose content but of low sperm density. In fact, some of our 
highest values for fructose so far recorded, were encountered in 
the semen of vasectomized, and thus completely azoospermic, 

Fructose and Fructolysis 139 

Seminal fructose as an indicator of male sex hormone activity; the 
'fructose test^ and its application to certain problems of sex 

The 'fructose test', originally described by Mann and Parsons 
(1947) and subsequently developed by Mann, Davies and Hum- 
phrey (1949), Mann, Lutwak-Mann and Price (1948) and Mann 
and Parsons (1950), is founded on the observation that the capacity 
of the accessory organs to produce fructose and, thereby, the actual 
level of fructose in the seminal plasma, reflects in a faithful manner 
the degree of testicular hormone activity in the male, and in this 
way provides an accurate indicator of endocrine testicular function. 
In experiments on rats and rabbits it was shown that seminal fruc- 
tose disappears almost completely within two weeks after castration 
and also that the postcastrate fall in the level of fructose can be 
prevented, or, if already developed, fully restored, by the implan- 
tation of testosterone (Fig. 12). The effect is not limited to labora- 
tory animals and analogous results were obtained with domestic 
animals such as the bull. 

The test can be carried out in two ways, by the chemical analy- 
sis of the seminal fluid collected from an intact animal by means 
of an artificial vagina, or by the analysis of accessory organs of 
reproduction obtained from the experimental animal by dissection. 
The first method gives an opportunity to observe in the same animal 
the time-sequence of changes brought about by castration and hor- 
monal treatment, and eliminates the sacrifice of the experimental 
animal. In the second procedure, on the other hand, the test can 
be used for a quantitative assay of male sex hormone activity in the 
whole body, isolated tissues, body fluids and hormone preparations; 
as an illustration. Fig. 13 gives a dosage-response curve obtained 
with coagulating glands of castrated rats which were injected for 
three weeks with known doses of testosterone propionate; following 
the last day of injections the rats were sacrificed, the coagulating 
glands dissected and used for fructose analysis. 

Below are discussed some of the endocrinological problems to 
which an approach was made in recent years with the aid of the 
'fructose test', applied either alone or in conjunction with the 'citric 
acid test', which depends on the relationship between the secretion 


The Biochemistry of Semen 

of citric acid by some of the accesory organs and the male sex 
hormone activity. 


Pellet implanted 

Pellet removed 

2 3 4 5 6 7 8 
Weeks after castration 
Fig. 12. Post-castrate fall and testosterone-induced rise of seminal fructose 
in rabbit; pellet: 100 mg. testosterone. 

(Mann & Parsons, 1947) 

{a) Time relationship between the onset of secretory activity in male 
accessory glands and spermatogenesis. In young rabbits (Davies and 
Mann, 1941 b), rats (Mann, Lutwak-Mann and Price, 1948), bull- 
calves (Mann, Davies and Humphrey, 1949) and boars (Mann, 1954), 

Fructose and Fructolysis 


fructose and citric acid appear in the accessory glands at an early 
age, before there is any evidence of active spermatogenesis; since 
the secretion of both these substances depends upon the presence of 
the male sex hormone, it must be concluded that the hormone 
begins to function in the male body well in advance of the actual 
spermatogenesis. Thus, for instance, in bull-calves appreciable 

5 25 50 

Daily dose of testosterone propionate (/ig.) 

Fig. 13. Dosage-response curves of testosterone propionate, using the coagu- 
lating glands of the rat; -O, fructose (/^tg.); • •, 

weight of organs (mg.). 

(Mann & Parsons, 1950) 

amounts of fructose are found in the vesicular secretion already at 
the age of about four months, whereas the first mature spermatozoa 
appear nearly eight months later. One cannot, of course, rule out 
the possibility that the testicular hormone is active in the bull-calf 
even before the age of four months, but if so, then either its con- 
centration is too small to produce a distinct response in the accessory 
organs, or else its action is countered by some other factors. 
ib) Effect of testosterone on the appearance of fructose in castrated 

142 The Biochemistry of Semen 

animals. The following experiment was carried out by Mann, Davies 
and Humphrey (1949) at the Agricultural Research Council Field 
Station at Compton, in Berkshire. Six bull-calves were used. These 
were castrated when one to two weeks old, i.e. at an age prior to the 
appearance of fructose in the seminal glands. Seven months later 
two of the castrated calves received subcutaneous implants of 0-5 g. 
pellets of pure testosterone, whereas the remaining four were left 
untreated. After another four weeks all six animals were sacrificed 
and their seminal glands dissected out, weighed, and examined both 
chemically and histologically. The unused portions of the hormone 
pellets were recovered from the subcutaneous tissue of the two 
hormone-treated calves; their weights were 0-344 and 0-338 g. 
respectively, showing that the quantities of testosterone absorbed 
per month per animal were 0- 1 56 and 0- 1 62 g., respectively. Chemical 
analysis revealed the presence of considerable amounts of fructose 
in the seminal glands in response to the four weeks' hormone treat- 
ment (51 mg. fructose per 100 g. tissue or 5-3 mg. fructose per 
total gland), as against a negligible fructose content in the untreated 
castrates (8 mg. per 100 g. or 0-25 mg. per total gland). In compari- 
son with and in contrast to the marked chemical difference, the 
evidence for the functional recovery in the seminal glands, as 
assessed by the histological examination, was practically impercep- 
tible (Plate VI). In this way we were able to provide evidence that 
the early effects of testosterone treatment can be established far 
more convincingly by the large percentage-increase in the fructose 
content of the seminal gland secretion, than by means of histological 
methods which at this stage failed to show significant changes in the 
glandular tissue. 

An investigation concerned with the response to testosterone was 
also made by Rudolph and Samuels (1949) on rats, and by Gassner 
and his co-workers (1952) on bulls. In castrated rats, a significant 
increase in the fructose content of accessory organs was noticed 
already ten hours after the injection of 1 mg. testosterone pro- 
pionate. In bulls, fructose disappeared from ejaculates within two 
weeks after castration but injections of testosterone propionate, if 
given within four weeks after castration, led to a rapid return of 
fructose production to the pre-castrate level; yet, in spite of the fully 
restored fructose level, such seminal plasma, when added to washed 













Histological sections from a tubule (mag. x 437), and the fructose content 
of seminal vesicle. 

A. from a bull-calf castrated when three weeks old, and killed when nine 

months old. 

B. from a bull-calf castrated when three weeks old, left untreated till 

eight months old, and then implanted with testosterone (0-5 g.); 
killed one month later, simultaneously with calf A. 

Fructose and Fructolysis 143 

spermatozoa obtained from a normal bull, was unable to support 
sperm metabolism to the same extent as plasma from normal i.e. 
non-castrated animals. 

An interesting example of the application of the fructose test to 
problems of infertility in man has been provided by a study of four 
eunuchoid patients who responded to androgenic treatment with a 
highly significant elevation of fructose in semen (Landau and 
Longhead, 1951). 

It seems probable that the fluctuations of fructose level in the 
semen of normal individuals may also be due, in part at least, to 
some periodic changes in the activity of the testicular hormone in 
the male body. Normal rats, injected with large doses of the male 
hormone invariably react by an increased level of fructose formation, 
well above that of non-treated controls. The effect is particularly 
striking with breeds of animals which exhibit a relatively low physio- 
logical level of fructose formation. In this connection, however, it is 
interesting to note that when injections of large doses of testosterone 
propionate into normal rats are continued to excess, e.g. 200 i-ig. 
daily for forty days, the state of overstimulation in the accessory 
organs is accompanied by a marked decline in the size of the testes; 
after seven weeks of such treatment the reduction in the weight of 
the testes is nearly 50% (Mann and Parsons, 1950). Injections of 
excessive doses of androgens are well known to produce harmful 
effects on the spermatogenesis in animals and in man (Moore, 1939; 
McCullagh and McGurl, 1939; Meckel, 1951). 

In normal bulls, a dose of 100 mg. testosterone propionate, 
repeated three times weekly for six weeks, appears to produce only 
a very slight increase in the level of fructose in semen (Gassner, 
Hill and Sulzberger, 1952). However, according to another report, 
sexual excitation prior to service has a stimulating effect on the out- 
put of fructose in bull semen (Branton, D'Arensbourg and Johnston, 

(c) Hormone-induced formation of fructose in subcutaneous trans- 
plants from accessory organs. Once the dependence of seminal fruc- 
tose upon the activity of the male sex hormone had been established 
it was possible to enquire into the mechanism of this hormonal rela- 
tionship. One of the problems to settle was the extent to which the 
process of fructose generation in accessory glands depends upon the 

144 The Biochemistry of Semen 

preservation of intact vascular and neural links. Insight into this 
matter was gained by the technique of subcutaneous transplantation, 
when it was demonstrated that small fragments of rat coagulating 
gland, about 1 mg. in weight, transplanted subcutaneously into 
normal adult male hosts, grew well and showed after some weeks 
of subcutaneous development a high content of fructose. Follow- 
ing castration of the hosts, the transplants lost their ability to 
form fructose but this was promptly restored by treatment with 
testosterone propionate. Perhaps the most remarkable fact in these 
experiments was that the growth of the grafts and their chemical 
secretory function occurred not only in male, but also in female 
hosts provided that the latter were treated with testosterone 
(Lutwak-Mann, Mann and Price, 1949). 

Thus, for the first time the effect of the male sex hormone on 
fructose secretion was demonstrated in tissue fragments dissected 
from the male accessory organs and developing in complete isola- 
tion from the rest of the male generative system. Actually, the trans- 
plants had an even higher fructose content than the corresponding 
intact glands of the graft-bearing hosts, because unlike intact glands, 
the grafts lack a secretory outlet. 

In another study, Price, Mann and Lutwak-Mann (1949, 1954) 
applied the transplantation technique, coupled with the chemical 
methods, to the problem of the androgenic activity of ovarian 
hormones in the female rat. Subcutaneous transplants of rat coagu- 
lating gland in female hosts were shown to secrete large quantities 
of fructose in response to injections of pregnant mare serum gona- 
dotrophin. A series of thirty injections of twenty international units 
of equine gonadotrophin was given daily; at autopsy the ovaries of 
the female hosts were enlarged at least tenfold and covered with 
numerous follicles and corpora lutea. In these rats, gonadotrophin, 
through a stimulating action on the ovaries, raised the output of 
ovarian androgens to an extent which induced the secretion of fruc- 
tose in transplants from the coagulating gland. 

{d) Effects of progesterone, stilboestrol and oestradiol. The nature 
of the ovarian androgen responsible for the formation of fructose 
is unknown, but there are indications that it may be related to 
progesterone or to a product of progesterone metabolism. An 
inquiry into the androgenic value of progesterone showed that large 

Fructose and Fructolysis 145 

doses of progesterone injected into castrated male rats have a 
definite androgenic effect; it was calculated that the androgenic 
value of 25 mg. pure progesterone is slightly more than that of 
0005 mg. testosterone propionate (Price, Mann and Lutwak-Mann, 
1949, 1954). 

Whereas progesterone exhibits some androgenic activity, stil- 
boestrol is endowed with the properties of an androgen-antagonist. 
The testosterone-catalysed secretion of fructose in the male accessory 
gland secretions of a rabbit can be suppressed very effectively by the 
subcutaneous implantation of 25 mg. stilboestrol (Parsons, 1950). 
In experiments in which a castrated rabbit received simultaneously 
implants of testosterone and stilboestrol, the latter prevented com- 
pletely the production of fructose by the accessory organs. 

It appears that in castrated bulls, small amounts of oestradiol 
dipropionate used together with testosterone, have a small but 
definite synergistic effect on the seminal vesicles and lead to a higher 
output of fructose in semen (Gassner, Hill and Sulzberger, 1952). 

Role of hypophysis 

The endocrine influence of the testes on the formation of fructose 
in accessory organs is integrated closely with the functioning of the 
anterior pituitary gland. Hypophysectomy, like gonadectomy, invari- 
ably results in a rapid decline in the level of fructose in the seminal 
plasma (Mann and Parsons, 1950). In the rabbit, for instance, a 
three to four weeks' period after castration or hypophysectomy alike, 
usually leads to complete disappearance of fructose so that an 
ejaculate collected by means of an artificial vagina three weeks 

Table 21. Ejfect of testosterone and pregnant mare serum 
gonadotrophin on the formation of fructose in rabbit prostate 

Weight of Fructose 

prostate content 

Rabbit (mg.) (//g. /organ) 

1. Normal 770 935 

2. 3 weeks after castration 280 20 

3. 6 weeks after castration and simultaneous 

implantation of testosterone (100 mg.) 1000 1220 

4. 4 weeks after hypophysectomy 149 10 

5. 6 weeks after hypophysectomy; for the last 

4 weeks injected 200 I.U. PMS-gonado- 

trophin 210 395 

146 The Biochemistry of Semen 

after the operation contains no more than 20 ^g. fructose, as com- 
pared with 500 to 1000 /^g., before the operation. Both castrated as 
well as hypophysectomized animals promptly respond to the sub- 
cutaneous implantation or injection of testosterone with renewed 
secretion of fructose. The same happens if instead of testosterone 
pregnant mare serum gonadotrophin is injected into a hypophy- 
sectomized animal (Table 21). 

The relationship between blood glucose and seminal fructose 

In addition to the hormones of the testis and the pituitary gland, 
yet another organ, the pancreas, exerts a profound influence upon 
the level of fructose in semen. The effect is an indirect one, and is 
brought about by the action of insulin on the level of blood glucose 
which in turn governs the level of fructose in semen. The existence 
of a causal link between the blood sugar level and seminal fructose 
was studied at first in animals with experimental diabetes; later, 
however, it was also shown in diabetic man (Mann and Parsons, 
1949, 1950). 

In rabbits, experimental diabetes can be produced with alloxan; 
best results are obtained by injecting intravenously into a rabbit 
75 mg. alloxan per kg. body weight, and repeating this dose one or 
two days later. Fig. 14 illustrates the course of such an experiment 
with a rabbit in which analyses of blood and semen were carried 
out regularly during a period of four months. At the outset of the 
experiment this animal had a blood glucose content of 100 mg. per 
100 ml., and about 600 i-ig. fructose per ejaculate or 70 mg. per 
100 ml. semen (fluid portion). However, following alloxan treatment, 
the rabbit developed severe glucosuria within two days and its blood 
glucose level rose to 350 mg./lOO ml.; at the same time, the level of 
fructose in semen began to increase, until three weeks later there 
was 500 mg. glucose per 100 ml. blood, and 3500 /^g. fructose per 
ejaculate or 320 mg. fructose per 100 ml. semen. When it was estab- 
lished that hyperglycaemia is followed by an increased concentra- 
tion of fructose in semen, the effect of insulin was examined and it 
was found that the insuUn-induced fall in blood glucose was followed 
by a reduction in the fructose content of semen; moreover, once the 
effect of insulin on blood glucose in the diabetic animal wore off, 
there was again an increase in seminal fructose. 

Fructose and Fructolysis 147 

Conditions similar to those in experimental diabetes seem to 
prevail also in man. In semen samples from diabetic patients we 

8 lO 12 


Fig. 14. Effect of diabetes and insulin on seminal fructose in rabbit. The 
period of insulin treatment is indicated by arrows. Semen was col- 
lected weekly. 

came upon fructose values which were well above the upper limit 
of normal variations; diabetic values ranged from 650 to 1230 mg. 
per 100 ml., and from 33-4 to 47-5 mg. per ejaculate. In a survey 
of 150 specimens of normal human semen, Harvey (1948) found 

148 The Biochemistry of Semen 

640 mg. per 100 ml. or 31-6 mg. per ejaculate to be the highest 
value. It is interesting to recall here that years ago Goldblatt (1935a) 
noticed a high reducing sugar value in human diabetic semen but 
attributed this to urinary glucose. 

Effect of malnutrition 

It has long been known that defective nutrition has a deleterious 
influence upon the male reproductive system. One of the earliest 
surveys of this problem is found in the monograph by Jackson (1 925); 
this was followed by the work on degenerative changes in testes and 
sterility associated with vitamin A and E deficiency and in later 
years, by many other nutritional studies which helped to accumulate 
a wealth of information on this subject, fully reviewed on several 
occasions (Asdell, 1949; Burrows, 1949; Lutwak-Mann, 1951; 
Mason, 1949; Reid, 1949; Russell, 1948; Samuels, 1948; Walton, 
1949). Most investigators in this field, however, particularly those 
concerned with problems of human fertility, were much more in- 
terested in the spermatogenic activity of the testicular tissue than 
in the function of the accessory organs of reproduction. It was, 
therefore, something of a departure when Moore and Samuels 
(1931) came forward with the demonstration that a few weeks of a 
diet deficient in vitamin B, or a quantitatively inadequate diet con- 
taining vitamin B, caused in male rats regressive changes in the 
accessory organs which, however, could be counteracted by the 
administration of testicular hormone or anterior pituitary extracts. 
They concluded that the primary lesion due to inadequate feeding 
was located in the pituitary gland and that as a result of the di- 
minished hypophyseal activity the testes received insufficient gona- 
dotrophic stimulus and were consequently, unable to produce the 
male sex hormone required for normal functioning of the accessory 
glands. A similar state of 'pseudo-hypophysectomy' was described 
by Mulinos and Pomerantz (1941) in rats as the result of a diet 
which was qualitatively adequate but halved in quantity; further 
supporting evidence was later provided by several groups of 
investigators (Pazos and Huggins, 1945; Goldsmith and Nigrelli, 
1950; Grayhack and Scott, 1952). In certain animal species sperma- 
togenesis was also shown to be affected by a vitamin B-deficient 

Fructose and Fructolysis 149 

diet (Marrian and Parkes, 1928; Dunn, Morris and Dubnik, 1947; 
Elson and Koller, 1948). 

Lutwak-Mann and Mann (1950a, b, 1951) applied chemical 
methods to the study of changes brought about in the secretory 
function of rat accesory organs by vitamin B-deficiency and inani- 
tion, and found that in rats maintained for four weeks on a deficient 
diet the content of fructose and citric acid in the accessory glands 
was reduced to a castrate level. By treatment with testosterone pro- 
pionate (0-2 mg. daily for one week) or with chorionic gonadotro- 
phin (200 units every other day for two weeks), the secretory activity 
of the glands could be completely restored. A further example of the 
effect of an unbalanced diet on the process of fructose secretion was 
provided by Lutwak-Mann (1951) who found that a diet with exces- 
sive fat content, even though not protein- or vitamin-deficient, also 
caused regression in rat accessory organs. Mann and Walton (1953) 
made a study of the effect of underfeeding on the genital functions 
in the bull and found that, in contrast to the testes, the secretory 
function of the male accessory glands was markedly affected by 
underfeeding. The concentration of fructose and citric acid in the 
semen of the underfed bull decreased by 30% and 60% respectively, 
of the original levels. In the bull, however, unlike in the rat, the 
effects of malnutrition as well as the recovery after the animal has 
been transferred back to its normal diet, developed comparatively 

The enzymic mechanism of fructose formation 

The experimental evidence available at present brought out the 
essential, though as yet not fully understood, role of the testicular 
hormone in the formation of fructose by the secretory apparatus 
of the male accessory glands, and indicated that blood glucose is 
the precursor of seminal fructose. Further details of the mechanism 
whereby glucose is converted in the accessory gland tissue to fruc- 
tose, were obtained from in vitro experiments; these showed that 
small amounts of fructose are formed as a result of incubation of 
minced accessory gland tissues with glucose, and that these tissues 
possess the entire enzymic system which can convert glucose to 
fructose (Mann and Lutwak-Mann, 1948, \95\a, b). 

It is an estabUshed fact that certain phosphorylated derivatives of 

1 50 The Biochemistry of Semen 

fructose, such as 6-phosphofructofuranose (Neuberg ester) and 1 : 6- 
diphosphofructofuranose (Harden- Young ester), are formed as in- 
termediary substances in the normal carbohydrate metaboUsm of 
muscle, liver and other animal organs. However, in the majority 
of animal tissues these phosphofructoses do not yield free fructose 
but are metabolized further to form pyruvic acid and lactic acid. 
In the semen, however, there are present in high concentration 
enzymes which belong to the group of phosphatases and include 
the 'alkaline' phosphatase; the latter capable of splitting a number 
of phosphohexoses, including 6-phosphofructose, 1-phosphofructose 
and 1 : 6-diphosphofructose, to phosphoric acid and free fructose. 
The alkaline phosphatase found in semen is derived from several 
accessory organs of reproduction but its principal source in higher 
animals is the seminal vesicle. Owing to this fact, the usual channels 
of carbohydrate metabolism are diverted in the vesicular tissue: 
phosphofructoses are not metabolized to lactic acid, as would be 
the case e.g. in muscle, but are dephosphorylated instead, so that 
free fructose is formed. 

In extracts made from bull seminal vesicle tissue it is possible to 
demonstrate the following reactions. When 1-phosphoglucose is 
incubated, 6-phosphoglucose is formed through the action of phos- 
phoglucomutase; next, part of 6-phosphoglucose is converted by 
phosphohexose isomerase into 6-phosphofructose. The equilibrium 
mixture of the two 6-phosphohexoses is acted upon by phosphatase 
and yields a mixture of free glucose and free fructose. Phosphohexose 
isomerase, together with alkaline phosphatase is also present in the 
seminal vesicle secretion and seminal plasma. Ram seminal plasma 
in particular, is a rich source of phosphatase active towards phos- 
phorylated sugars (Table 17, p. 120). 

The fact that whereas a mixture of glucose and fructose is the 
result of the phosphatase activity in the glandular tissue, yet, only 
one sugar, that is fructose, accumulates in the secretion, may have 
its explanation in a more effective re-utilization of glucose than 
fructose, by the glandular tissue itself. Thus, we have found that 
tissue slices from the rat coagulating gland can glycolyse anaero- 
bically glucose at a much higher rate than fructose; this, in 
turn, may be due to the ability of the tissue to re-phosphorylate 
more effectively glucose to 6-phosphoglucose, than fructose to 

Fructose and Fructolysis 151 

6-phosphofructose. Such evidence as is at present available, derived 
from both in vivo and /// vitro experiments, indicates that the 
enzymic reactions involved in the conversion of blood glucose to 
seminal fructose are as follows. 

Blood glucose 


^ Phosphorylase 


\ Phosphoglucomutase Phosphohexose 

6-Phosphoglucose \ >'6-Phosphofructose 

f \ 

Glucokinase Alkaline phosphatase 



Glucose ^^ Seminal fructose 

Anaerobic and aerobic utilization of carbohydrate by spermatozoa 

The spermatozoa of the sea-urchin and certain other animals 
derive their energy for movement chiefly from respiratory processes; 
in contrast, the survival and motility of sperm ejaculated by animals 
with internal fertilization, such as mammals, is possible for most 
of them also in absence of oxygen, provided that the sperm cells 
remain in contact with seminal plasma. Mammalian spermatozoa 
possess only a negligible reserve of intracellular glycogen and depend 
therefore, under anaerobic conditions, on an extracellular source of 
energy. In species which contain fructose as a normal constituent 
of the seminal plasma, anaerobic fructolysis is the metabolic process 
which enables the spermatozoa to survive without oxygen. Should, 
however, the spermatozoa become separated from the seminal 
plasma by centrifugation and washing, they could not carry on 
anaerobically unless the seminal plasma were restored or replaced 
by glycolysable carbohydrate. 

The stimulating effect of pure sugars on sperm motility has been 
noticed by some of the early investigators of semen. In 1931 Ivanov 
observed that dog spermatozoa suspended in an isotonic solution of 
glucose and phosphate retained their motility when the respiration 
had been abolished either by poisoning with cyanide or by replace- 
ment of oxygen with hydrogen. This observation is of particular 
interest in view of the fact that the dog has no seminal vesicles and 

152 The Biochemistry of Semen 

no fructose in the seminal plasma; the possibility, of course, must 
not be overlooked that there may be in dog semen some other sub- 
stance of nutrient value to the spermatozoa. Redenz (1933) has 
shown that bull spermatozoa glycolyse glucose, fructose, and man- 
nose to lactic acid, and that the presence of these sugars, but not 
that of sucrose, lactose, or glycogen, is beneficial to sperm motility. 
His findings were confirmed by others and it has since become 
an established fact that the metabolism of spermatozoa in several 
mammalian species including man, ram and bull, is predominantly 
of a glycolytic character (Ivanov, 1935; Shergin, 1937; Comstock, 
1939; MacLeod, 1939, 19436; Lardy and Phillips, \9A\a\ Moore and 
Mayer, 1941; Henle and Zittle, 1942; Ross, Miller and Kurzrok, 
1941; Salisbury, 1946). 

Sperrnatozoa obtained directly from the epididymis of a bull, 
ram, or boar, resemble washed ejaculated sperm in that they are 
incapable of survival under purely anaerobic conditions. While in 
the epididymis, the spermatozoa have no access to fructose and are 
immotile; the onset of motiUty coincides with their passage along 
the male genital tract and contact with the seminal plasma. The acti- 
vating influence of fructose on previously immotile spermatozoa 
can be convincingly demonstrated in a simple manner. Fresh epi- 
didymal spermatozoa are suspended in bicarbonate-Ringer solution; 
two 'hanging drops', one of the suspension and another, a little 
further away, containing a 1 % solution of fructose in bicarbonate- 
Ringer solution, are placed on the underside of a cover-sUp; to 
observe the motility of the sperm under the microscope, the cover- 
slip is fixed to the top of a small glass chamber in which one can 
create anaerobic or aerobic conditions by passing through the cham- 
ber a gas mixture of 95% N2-5% CO2 or 95% 02-5% CO2. In the 
absence of oxygen, the spermatozoa can be seen to be almost com- 
pletely immotile, but when the two drops are brought together the 
sperm movement begins and continues for a long time. Aerobically, 
the effect of fructose is less striking because oxygen induces endo- 
genous respiration and this in itself provokes motility in epididymal 
spermatozoa. However, even in the presence of oxygen, fructose 
still has some influence owing to the process of aerobic fructolysis. 

Under anaerobic conditions, the final product of sperm fructolysis, 
lactic acid, cannot be oxidized further. In the presence of oxygen, 

Fructose and Fructofysis 153 

however, the situation differs in that the rate of fructose utiliza- 
tion becomes smaller, and moreover, lactic acid undergoes further 
oxidation, thus providing an additional source of metabolic energy. 
It remains for further study to ascertain what type of carbohydrate 
metabolism predominates in spermatozoa during their existence in 
either the male or female genital tract. However, so far as in vitro 
studies are concerned, they show that lactic acid can be efficiently 
oxidized by spermatozoa even when the partial pressure of oxygen 
has been reduced to a level as low as that which normally prevails 
in animal tissues (Mann, 1951Z)); suitably diluted suspensions of ram 
spermatozoa show in presence of 1 % O2 a respiratory rate as high 
as in air, and lactate is capable of maintaining the oxygen uptake 
equally well in 1% as in 20%, oxygen. 

Pasteur effect and the ^metabolic regulator'' 

It was said earher that the spermatozoa obtained directly from 
the epididymis in some ways behave like suspensions of washed 
ejaculated sperm; as a matter of fact, however, these two types of 
sperm cells possess distinct characteristics (Henle and Zittle, 1942; 
Lardy, Hansen and Phillips, 1945). Washed epididymal bovine 
spermatozoa have a lower endogenous respiration than those in 
ejaculated bull semen. But if sugar is added or if the spermatozoa 
are removed from the epididymis after a period of storage in the 
refrigerator, then their oxygen uptake is distinctly higher. More- 
over, on addition of sugar, epididymal spermatozoa produce lactic 
acid much more rapidly under anaerobic than aerobic conditions, 
whereas in ejaculated sperm the rate of glycolysis is not much 
higher in the presence than in the absence of oxygen. To account 
for these differences, the Wisconsin workers determined the rate of 
anaerobic and aerobic glycolysis as well as of oxygen uptake, in 
epididymal spermatozoa to which glucose was added, and calcu- 
lated the 'Meyerhof oxidation quotient' which measures the Pasteur 
effect, that is the extent to which glycolysis is inhibited by oxygen. 
The average value for the Meyerhof quotient calculated from twelve 
experiments on bull epididymal sperm was 9-6, as against 5 recorded 
for ejaculated sperm (Lardy, 1952). This difference, according to 
Lardy, Ghosh and Plant (1949), is due to the presence in bull sper- 
matozoa of a 'metabolic regulator' which occurs in the epididymal 

1 54 The Biochemistry of Semen 

sperm in a 'bound form' but is released in an 'active form' after 
ejaculation. Continuing their study, these workers observed that 
heat-inactivated bull semen, or semen and testicular extracts heated 
with sodium hydroxide, increased the rate of aerobic fermentation 
of sugars by baker's yeast, without affecting markedly yeast respi- 
ration or anaerobic fermentation. The yeast-stimulating factor was 
extracted with carbon tetrachloride from alkaline hydrolysates of 
hog testes and obtained in the form of yellow coloured crystals 
which proved to be elementary sulphur (Ghosh and Lardy, 1952). 
Yeast reduces sulphur to HgS which is probably the agent ultimately 
responsible for the stimulation of the aerobic fermentation. The 
identity of the yeast factor with sulphur was verified by reproducing 
the stimulating effect on yeast with pure rhombic sulphur. Sulphur 
as such, however, cannot be the sperm 'regulator' since it is without 
influence on the Pasteur effect in epididymal spermatozoa. On the 
other hand, a number of sulphydryl compounds such as cysteine, 
reduced glutathione and hydrogen sulphide have been found to 
stimulate the respiration and aerobic glycolysis of epididymal 
sperm and the possibility remains, that the 'metabolic regulator' 
is, in fact, a sulphydryl compound, liberated during ejaculation 
from the spermatozoa, with a sulphydryl group in labile form, which 
can be easily removed and oxidized to sulphur by alkaline hydrolysis. 
The peculiar changes in the metabolic properties of spermatozoa 
during cold-storage of the epididymis, are equally in need of eluci- 
dation. A problem which also deserves further biochemical study 
is the 'ripening' phenomenon which takes place in the spermatozoa 
while they remain in the epididymis. Presumably, the metabolism 
of sperm in the epididymis is related in some as yet unknown 
manner to the structural changes associated with sperm maturation 
processes, such as the migration of the 'kinoplasmic droplet'. 

Intermediary reactions in sperm fructo lysis and the role of phosphorus- 
containing coenzymes 
The ability of washed spermatozoa to convert into lactic acid 
equally well added fructose, glucose and mannose is due in all 
probability to the fact that the metabolic degradation of these 
three sugars is initiated by the sam.e hexokinase-catalysed reaction 
with adenosine triphosphate. 

Fructose and Fructolysis 155 

Adenosine triphosphate (ATP, formula in Fig. 15) represents an 
intracellular constituent and a coenzyme of considerable importance 
in the economy of the sperm cell. An observation that a considerable 
proportion of the acid-soluble phosphorus in bull spermatozoa 
yields orthophosphate after 7 min. hydrolysis with n-HCI first 
suggested the presence of ATP (Lardy and Phillips, 1945). In the 
same year, the readily-hydrolysable phosphorus compound was 
isolated from ram spermatozoa and its identity with ATP estab- 
lished by chemical analysis (Mann, 1945«, c); the content of ATP 
in ram spermatozoa is 2-6-6-6 mg. labile phosphorus or 0-6-1 -5 mg. 
of adenine amino-nitrogen per 100 ml. semen (see also Table 16). 
The occurrence of ATP in ram and boar spermatozoa has also been 
confirmed by Ivanov, Kassavina and Fomenko (1946) who found 
that the phosphorus compound which they purified from sperm 
induced contractions of muscle actomyosin threads in the same 
manner as ATP isolated from skeletal muscle. ATP was also found 
in sea-urchin spermatozoa (Rothschild and Mann, 1950), the con- 
centration of ATP in the semen of Echinus escidentus resembling 
that found in the ram. 

Spermatozoa, even after they have been repeatedly washed so as 
to remove the phosphatases present in seminal plasma, continue to 
exhibit a high phosphatase activity against ATP, and all evidence 
available at present points to sperm ATP-ase as the enzyme which 
is directly responsible for the supply of energy essential for normal 
motihty and survival of the sperm cell. The losses due to utilization 
of ATP are made good by re-synthesis which takes place during the 
normal metabolism of spermatozoa, and any interference with inter- 
mediary enzymic reactions which renders the sperm cell incapable 
of breaking down or building up ATP, leads to a decrease in both 
metabolism and motility. Using ram spermatozoa as experimental 
material under a variety of conditions, we have found that a diminu- 
tion in the content of ATP invariably coincides with impaired sperm 
motility (Mann, 1945^7, b, c). Thus, for instance, in ram spermatozoa 
deprived of the fructose-containing seminal plasma by washing, 
ATP content as well as motility went down simultaneously on 
anaerobic incubation, but both ATP and motility could be 
preserved anaerobically in sperm suspensions provided with 
glycoly sable material. 


The Biochemistry of Semen 

The activity of hexokinase, the enzyme which brings about the 
initial reaction between ATP and glycolysable sugar, can be demon- 
strated directly in washed spermatozoa (Mann, 1945/)). If we add 
to a ram sperm suspension sugar (glucose, fructose or mannose), 



Spermatozoa Seminal Plasma 

rifc— ^ 



1 A 



Diphosphofructose "J 

1 "li^"" 

^l' OH>— f OH 


Phosphotrjose i^ ^ 


1 D (-^Fructose 

^H^K ^^ 


1 ^N A 




Lactic acid 

AXR > 



Phosphopyruvic "^ ^^ 

acid y 



-< i 

Pyruvic acid 

L J 

Fig. 15. Diagrammatic representation offructolysis in semen. 

sodium fluoride, and ATP, there is on incubation a rapid disap- 
pearance of half of the readily-hydrolysable phosphorus of ATP 
and formation of adenosinediphosphate (ADP) and 6-phospho- 
hexose. Of course, without added ATP and fluoride, 6-phospho- 
hexose does not accumulate but the degradation of sugar continues 
uninterruptedly to its final stage, i.e. the formation of lactic acid. 

Fructose and Fructolysis 1 57 

If either glucose -alone or fructose alone is used as substrate, the 
rate of lactic acid production is the same (Mann and Lutwak-Mann, 
1948), but if washed spermatozoa are made to act on a 1 : 1 mixture 
of glucose and fructose, then the rate of fructose utihzation becomes 
much less than 50% (Mann, \95\b). This 'sparing effect' of glucose 
on the utilization of fructose is probably due to the competitive 
inhibition of sperm hexokinase. Slein, Cori and Cori (1950) have 
shown that when brain or yeast hexokinase acts upon ATP and on 
an equimolar mixture of glucose and fructose, the aldosugar is phos- 
phorylated much more rapidly than the ketosugar. Under natural 
conditions only fructose is present in whole semen, but not glucose, 
so that the possibility of the latter interfering with fructolysis does 
not arise. But there is preferential utilization of glucose in the acces- 
sory glands, directly responsible for the accumulation of fructose, 
and this may be due to a stronger affinity of hexokinase for glucose 
than for fructose. A competition for hexokinase, between glucose 
and fructose, is also consistent with the observation that in bull 
semen incubated with an egg-yolk-diluent, the initial rate of fructo- 
lysis is temporarily retarded (Vantienhoven, Salisbury, VanDemark 
and Hansen, 1952), as is also the case in semen incubated with cow 
follicular fluid (Lutwak-Mann, 1954); both egg-yolk and follicular 
fluid contain glucose. 

The phosphohexose formed from fructose as a result of hexo- 
kinase activity is 6-phosphofructofuranose; in the case of glucose 
the product is 6-phosphoglucopyranose. The latter, however, is 
readily converted to 6-phosphofructose by phosphohexose iso- 
merase, and from this stage onwards, the enzymic degradation of 
glucose and fructose is identical. The chain of events which in whole 
semen leads from fructose to lactic acid, is diagrammatically depicted 
in Fig. 15. In the normal course of fructolysis, 6-phosphofructose is 
phosphorylated by ATP in a reaction catalysed by phosphofruc- 
tokinase, to yield 1 : 6-diphosphofructose and ADP; disphospho- 
fructose is next split by zymohexase into two molecules of phospho- 
triose. Like the action of phosphofructokinase, that of zymohexase 
was demonstrated directly in spermatozoa (Mann, 1945^). 

The subsequent steps in sperm fructolysis are analogous to the 
corresponding phases in muscle glycogenolysis and blood glucolysis, 
and involve the participation of cozymase (diphosphopyridine 

158 The Biochemistry of Semen 

nucleotide) which as Winberg (1941) showed, is a characteristic 
intracellular constituent of spermatozoa. The cozymase-catalysed 
phase of fructolysis consists of two closely interwoven oxidoreduc- 
tion processes (Mann, 19456; Mann and Lutwak-Mann, 1947). 


_,, , ^ . (, Phosphotriose dehydrogenase ) t^, i , • • j 

Phosphotriose — ^^ . ^-^ Phosphoglyceric acid 


H2 NaF inhibits 

\ Y 

Cozymase Phosphopyruvic acid 


Ha cozymase 


Lactic acid^ ; Pyruvic acid 

Lactic dehydrogenase -^ 

The first oxidoreduction involves the oxidation of phosphotriose 
to phosphoglyceric acid by phosphotriose dehydrogenase, and a 
simultaneous reduction of cozymase to dihydrocozymase; the oxida- 
tion of phosphotriose is coupled with an esterification of inorganic 
phosphate and the synthesis of ATP; the oxidation product, phos- 
phoglyceric acid, is converted by enolase to phosphopyruvic acid, 
the phosphate of which is transferred to ADP, thus producing 
pyruvic acid and ATP. The second oxidoreduction is between 
dihydrocozymase and pyruvic acid: dihydrocozymase is oxidized to 
cozymase, and pyruvic acid is reduced by lactic dehydrogenase to 
L(+)-lactic acid. When washed ram spermatozoa are treated with 
fluoride (to inhibit enolase), and incubated with added phospho- 
triose and pyruvate, the two oxidoreduction processes continue as 
usual but in addition to lactic acid there is an accumulation of 
phosphoglyceric acid. lodoacetate, on the other hand, abolishes the 
oxidoreductions in washed sperm and thus deprives them of the 
ability to produce lactic acid. 

The not unimpressive array of facts available from the outlined 
studies on spermatozoa strengthens the belief that ATP is the car- 
dinal link between the activity of spermatozoa on the one hand, 
and carbohydrate metabolism on the other. In whole semen, ATP 
acts continually as phosphate-donor and acceptor in the course of 
fructolysis. The content of ATP and with it the motility of ejaculated 

Fructose and Fructolysis 1 59 

spermatozoa, both depend on the maintenance of the normal 
metaboHsm of fructose. 

We still remain confronted with two questions to which, it is 
confidently hoped, further research will bring answers. One involves 
the as yet obscure position in the semen of animal species which lack 
fructose. Secondly, one wonders why nature should have chosen 
fructose and not glucose, as the natural substrate for sperm meta- 
bolism. At this point, conditions in another body fluid, the milk, 
come to mind; there, the occurrence of lactose poses a somewhat 
similar question. But in considering the matter, several facts must 
be taken into account. To begin with, if glucose and not fructose 
were present in semen, its concentration could hardly be expected 
to exceed that of blood and other body fluids. Thus, it might not 
be sufficient to satisfy the metabolic requirements of spermatozoa 
which, unlike most other animal cells, are capable of utilizing fruc- 
tose anaerobically; it is worth noting that on the whole, yeasts and 
bacteria are also unable to consume fructose at the same rate as 
glucose. Presumably, this enables the spermatozoa to draw freely 
upon seminal fructose without, as it were, competition from other 
tissues. Lastly, the intimate relationship between seminal fructose 
and the male sex hormone must not be lost sight of; it would be 
rather difficult to envisage a similar dependence in the case of 
glucose, bearing in mind the ubiquitous occurrence and physio- 
logical function of this sugar. 


Spermine, Choline, Ergothioneine , and 
certain other Bases in Semen 

Spermine. Occurrence of crystalline spermine in human semen; its 
chemical nature and properties. Derivatives of spermine and their use 
in forensic medicine. Synthesis of sperrnine. Spermidine. Oxidation of 
spermine and spermidine by diamine oxidase. State of spermine in semen. 

Choline. The Florence reaction in semen. Enzymic liberation of choline 
from precursors in semen. Phosphorylcholine and glycerylphosphoryl- 
choHne. Physiological function of free and bound choline. Choline 

Ergothioneine. Isolation of ergothioneine from the boar seminal vesicle 
secretion. The function of seminal ergothioneine and its behaviour towards 
sulphydryl-binding substances. Biogenesis of ergothioneine. 

Creatine and creatinine. Occurrence in mammalian semen, and in the 
sperm and gonads of invertebrates. Phosphocreatine and phosphoarginine. 

Adrenaline and noradrenaline . Occurrence in semen and accessory organs. 
Enzymic oxidation. Pharmacodynamic properties. 

Among the chemical characteristics which distinguish semen from 
other tissues and body fluids is the occurrence of certain nitrogenous 
bases, largely betaines, which are rarely found elsewhere in the 
animal body. Of these, spermine is the oldest-known, and ergo- 
thioneine the most recently discovered. 


Occurrence of crystalline spermine; its chemical nature and properties 
When human semen 'had stood a little while, some three-sided 
bodies were seen in it, terminating at either end in a point; some 
were of the length of the smallest grain of sand, and some were a 
little bigger, as in Fig. A. They were further as bright and clear as if 
they had been crystals.' Thus, in a letter of November 1677, addressed 
to the Royal Society, Antoni van Leeuwenhoek reported the dis- 
covery of the crystalline substance in semen which later became 



ditld materia pauallumtempoTiiJieterAt^ in ea, ohjtrvabsrjtur tri- 
idteraiesfi^ur^ ^b utraque pirte tn acttieum defin'r:tis ^ quthuf- 
dim icngitudfi minuttfiiwd' arena^ aliqu£ ^ltqt4ihtH urn wajores^ 
A f^ l\ % «^ fij* A. Frateredy adeo ntttd£ &o ptUucida^ acfi 

" i| V ^' cr\fiallin£ juijjent. 


Crystals in human semen as seen (from top to bottom) by Leeuwenhoek 
(1677), Fuerbringer (1881) and Poehl (1898). 

Spermine, Choline, Ergothioneine 161 

known as spermine. This was actually the letter in which Leeuwen- 
hoek also communicated for the first time the discovery of living 
spermatozoa and their movement in fresh semen; it was published 
the following year in the Philosophical Transactions (Plate VII). 
During the 200 years which followed, the same crystalline substance 
was rediscovered by several investigators, most of whom were 
apparently unaware of either Leeuwenhoek's original, or of the 
others' later observations. 

Vauquelin(1791) observed in a semen sample which he left standing 
for four days, the deposition of 'cristaux transparens, d'environ 
une ligned de long, tres-minces, & qui se croisent souvent de maniere 
a representer les rayons d'une roue. Ces cristaux isoles nous ont 
offert, a Faide d'un verre grossissant, la forme d'un solide a quatre 
pans, termines par des pyramides tres-allongees, a quatre faces.' 
After having studied the properties and behaviour of these crystals 
towards different solvents, Vauquelin concluded that 'la nature de 
ces cristaux est analogue a celle du phosphate de chaux ou la bas 
des os'. The belief that the sperm crystals consist of ordinary phos- 
phate persisted throughout the best part of the next century. In the 
meantime, however, the same crystalline substance was found out- 
side the semen in other tissues and body fluids, including sputum, 
leucaemic blood, liver, spleen and old pathological-anatomical pre- 
parations, so that towards the close of the XlXth century spermine 
was already known by no less than ten names of various distin- 
guished clinicians, anatomists and physiologists, including, in 
chronological order, Charcot, 1853; Foerster, 1859; Harting, 1859; 
White, 1861 ('leucosine'); Friedrich, 1864; Huppert, 1864; Boettcher, 
1865; Neumann, 1866; Eberth, 1869; Ley den, 1872; and Zenker, 
1876. But in the end, the medical world at large restricted itself 
largely to the use of two names, 'Charcot-Leyden crystals' with 
reference to organs and sputum ('asthma crystals'), and 'Boettcher 
crystals' with reference to semen. Boettcher himself preferred to 
call the substance 'Spermatin', and regarded it as a protein; he 
published his paper 'Farblose Krystalle eines eiweissartigen Korpers 
aus dem menschlichen Sperma dargestellt' in 1865, without however, 
taking the trouble to mention the previous investigators. 

The credit for having been the first to recognize spermine as the 
phosphate of a new organic base, is due to Schreiner (1878) who 

162 The Biochemistry of Semen 

succeeded in preparing a number of derivatives of spermine includ- 
ing the hydrochloride, but who unfortunately deduced from his 
analyses the wrong formula for the base, C2H5N. In consequence of 
this, spermine was confused with ethyleneamine, CoH^NH, and with 
piperazine. For years to follow, piperazine was offered by a large 
pharmaceutical firm in Berlin under the trade name of 'Spermin', 
and as late as 1903 the formula of piperazine appeared under the 
name of spermine in Thierfelder's Hoppe-Seyler' s Handbuch der 
chemischen Analyse. A great advocate of the manifold curative pro- 
perties of 'real' spermine, i.e. as isolated from human semen, bull 
testes, or other organs, was Alexander von Poehl, who believed in the 
'action of spermine as a physiological tonic on auto-intoxications' 
(1893), and who is best known for the monograph Die physiologisch- 
chemischen Grundlagen der Spermintheorie which he published in 
St. Petersburg in 1898. Poehl's book contains the records of numer- 
ous cases ranging from scurvy to syphilis, treated, apparently suc- 
cessfully, with the 'Sperminum Poehl'. His pharmacological and 
clinical work aroused much controversy, was subjected to severe 
criticism, and was finally altogether rejected. Yet, it is not entirely 
improbable that there is some justification for Poehl's 'spermine 
theory'. Apart from its general pharmacodynamic properties similar 
to those of other biological polyamines (Guggenheim, 1940), sper- 
mine may well possess some other, more specific pharmacological 
activity. Administered parenterally, spermine is known to be 
toxic to mice, rats and rabbits (Rosenthal, Fisher and Stohlman, 
1952). It has also been shown to possess bacteriostatic properties. 
The inhibition of the growth of Staphylococcus aureus by human 
seminal plasma can be attributed, according to Gurevitch and his 
colleagues (1951), to the high content of spermine in human semen. 
Another striking example of the growth-inhibiting action of sper- 
mine has been provided by Hirsch and Dubos (1952); following up 
an observation that the extraction of animal tissues with mixtures of 
water and ethanol yields material with tuberculostatic activity in 
vitro, these authors isolated from tissue extracts a crystalline anti- 
mycobacterial substance which they found to be identical with 
spermine phosphate. 

Leaving aside Poehl's pharmacological observations, one must 
nevertheless appreciate his contribution to the chemistry of spermine. 

Spermine, Choline, Ergothioneine 163 

Not only was he able to refute the mistaken belief in the identity 
of spermine and diethylenediamine but he was also the first 
to analyse correctly the gold salt and the chloroplatinate of sper- 
mine and to establish that 'the organic base which is at the bottom 
of these double salts would have the composition, C5H14N2 (1898, 

However, not until 1924 was conclusive chemical and crystallo- 
graphic evidence brought forward to prove the identity of spermine 
isolated from semen with the base obtained by similar methods from 
various animal organs and also from yeast. Credit for this is due to 
Otto Rosenheim (1924). In 1924, Dudley, Mary Rosenheim and O. 
Rosenheim in England, and Wrede in Germany, concluded from 
the molecular weight estimations of benzoylspermine and m-mixo- 
benzoylspermine, respectively, that the molecular formula of sper- 
mine is C10H26N4, and not as formerly assumed C5H14N2. 

Rosenheim and his colleagues obtained spermine in the free state 
as a crystalline, optically inactive substance which melts between 
55° and 60°, and distills at about 150° in vacuo without decomposi- 
tion. They also found that the base is stable in hot concentrated 
alkali and in boiling hydrochloric acid. Their relatively simple 
method of isolation depends on the steam-distillation of spermine 
from a strongly alkaline solution. 

Derivatives of spermine and their use in forensic medicine 

There are several well-defined compounds of spermine, a list of 
which is given in Table 22. Apart from the highly characteristic 
insoluble phosphate, spermine can be identified particularly easily 
as a picrate, which can be prepared from the free base, the phos- 
phate, or directly from semen. Spermine picrate, like the phosphate, 
is extremely insoluble in water. Crystallographic analysis has shown 
that it is identical with the substance responsible for the so-called 
Barberio reaction, a chemical test of diagnostic value in forensic 
medicine. Barberio's (1905) test consists in the addition to semen, 
or to an aqueous extract from the seminal stain, of picric acid in 
concentrated aqueous or ethanolic solution; in the presence of picric 
acid, there follows within a few minutes, the formation of abundant 
yellow crystals, resembling in shape the crystals of spermine phos- 
phate. The statement by Barberio that the reaction appears to be 


The Biochemistry of Semen 

specific for human semen as distinct from animal semen, has been 
corroborated by other ItaUan investigators, particularly by Baecchi 
(1912). In the experience of Littlejohn and Pirie (1908), Barberio's 
reaction is best carried out as follows; a small piece of the stained 
fabric is placed upon a glass slide and macerated in a drop or two 

Table 22. Chemical properties of spermine and its derivatives 

Compound Formula 

Free base C10H26N4 

Phosphate CioH26N4-2H3P04-6H20 

Hydro- CioH2eN4-4HC] 


Picrate CioH26N4-4C6H307Na 

Chloro- CioH26N4-4HCl-4AuCls 

Chloro- CioH26N4-2H2PtCl6 


Benzoyl CioHaeNi^COCeHs 

Tetra- CioH26N4-4CioH608N2S 



Needle-shaped, colourless, odour- 
less crystals; easily soluble in 
water, ethanol and butanol, in- 
soluble in ether, benzene and 
ligroin, m.p. 55-60°, m.w. 202 

Lenticular crystals from water, 
long needles from ethanol. Insol. 
in cold water, sol. 1 : 100 in boil- 
ing water, easily soluble in dilute 
acid or alkali, m.p. 240°, m.w. 
504. Identical with 'Boettcher's 
crystals' and 'Charcot-Leyden 

Short prismatic needles, extremely 
soluble in water, insol. in 
acetone, ether and chloroform, 
m.p. 300-310° 

Yellow needles, become black at 
242°, melt sharply with decom- 
position at 248-250° 

Golden-yellow, lustrous leaflets, 
m.p. 225° (Wrede 218°) 

Orange-yellow, well-formed cry- 
stals, m.p. 242-245° 

Lemon-yellow crystals, m.p. 288- 

Crystallizes from a solution in hot 
acetone on addition of ligroin, in 
woolly balls of fine needles, m.p. 
155°, m.w. 618 

CrystalliLes if flavianic acid is used 
in excess. On recrystallization 
from water the diflavianate is 

Spermine, Choline, Ergothioneine 165 

of distilled water; to the extract thus obtained (concentrated by 
evaporation, if necessary), a very small drop of an aqueous saturated 
solution of picric acid is added, by means of a platinum loop; after 
a minute or two a cover slip is applied, and the preparation examined 
under the microscope; 'when fully developed, the crystals have the 
form either of obtuse or sharp-ended needles, or of rhombic prisms 
frequently crossed by a refrangent line at their equator. Sometimes 
crosses are formed, and more rarely stars.' (For further particulars 
concerning Barberio's test see Harrison, 1932.) Another deriva- 
tive of spermine used in medico-legal laboratories is spermine 
flavianate; this crystalline compound forms the basis of Puranen's 
reaction (Puranen, 1936; Berg, 1948). 

Synthesis of spermine 

The final elucidation of the chemical structure of spermine was 
achieved in 1926. The existence of two chains A^ — C — C — C — A'^ 
and of one chain N—C — C — C — C — A^ in the spermine molecule 
was inferred by Dudley, Rosenheim and Starling from the identi- 
fication of pyrrolidine and tetramethyltrimethylene diamine as 
degradation products of spermine hydrochloride and decamethyl 
spermine, respectively. In Wrede's laboratory, the presence of two 
3C chains and one AC chain was established the same year as a 
result of studies on split products obtained from spermine by oxidation 
with molecular oxygen in presence of copper. The final proof was 
provided by the English investigators when they accomplished the 
synthesis of spermine and showed it to be, a,(5-bis [y'-amino- 


I ■ I II 


The oxidation of spermine gives rise to a volatile base associated 
with the characteristic odour of semen. The appearance of the 
semen-like odour during treatment of spermine chloroaurate with 
metallic magnesium, first described by Schreiner (1878), probably 
involves also an oxidation. The base is volatile in steam, and forms 
a crystalline hydrochloride and chloroaurate (m.p. 204-206°); it is 
probably identical with A^-y-aminopropylpyrroline. 

166 The Biochemistry of Semen 


Dudley, Rosenheim and Starling (1927) also succeeded in the 
isolation of spermidine, a base present in the mother-liquor after 
separation of spermine phosphate; spermidine phosphate is much 
more soluble than spermine phosphate and crystallizes from the 
25% ethanolic mother-liquor, after the removal of spermine phos- 
phate, when the concentration of alcohol is increased to 50%. The 
properties of spermidine are similar to those of spermine. It gives 
the same pyrrole reaction and behaves in an identical manner 
towards precipitating reagents including phosphotungstic acid. 
Spermidine is optically inactive, and yields like spermine, the semen- 
like odour when a solution of its chloroaurate is treated with 
magnesium. The structural formula of spermidine, proved by syn- 
thesis, is that of a-[y'-aminopropylamino]-(5-aminobutane: 


I I 

NHa NH2 


The close chemical relationship of spermine and spermidine 
suggests that the two bases may be related metabolically. Little, 
however, is as yet known about the biogenesis and metabolism of 
either of these two substances. So far as spermine in human semen 
is concerned, there can be little doubt that its high concentration 
which is of the order of 50-250 mg./lOO ml., is due chiefly to the 
prostatic secretion. Fuerbringer showed in 1881 that the prostatic 
gland contributes by far the greatest part of seminal spermine; on 
addition of two drops of 1 % solution of (NH4)2HP04 to ten drops 
of freshly collected prostatic fluid, Fuerbringer observed an almost 
instantaneous formation of Boettcher's crystals; the examination of 
secretions of the other accessory organs gave a negative result. The 
concentration of spermine in the human prostate is subject to varia- 
tions but exceeds that of any other organ. This follows both from 
Harrison's findings (1931) as well as from the survey carried out by 
Hamalainen (1947) who determined spermine as flavianate after pre- 
cipitation from trichloroacetic acid extracts; the highest values 
obtained by the Finnish investigator in the different organs (ex- 
pressed as mg. spermine phosphate per 100 g. tissue, wet weight) 

Spermine, Choline, Ergothioneine 167 

were, prostate 456, pancreas 77, adrenal 58, liver 43, spleen 40, 
testis 29, ovary 9. It is doubtful if Fuerbringer's (1886) belief in the 
'vitalizing' effect of the prostatic secretion upon spermatozoa could 
be applied to spermine as such; Harrison (1931, 1933) was unable to 
detect any activating influence of spermine phosphate on human 
spermatozoa. In contrast to human, bull semen contains no sper- 
mine; this is not surprising in view of the absence of a true functional 
prostate in the latter species. 

Oxidation of spermine and spermidine by diamine oxidase 

Spermine and spermidine, both undergo oxidation in the presence 
of diamine oxidase, an enzyme of which there is about a hundred 
times more in human seminal plasma than in blood serum (Zeller, 
1941); this finding together with observations by earlier investigators 
who found that the oxygen uptake of human semen is linked with 
the seminal plasma rather than the spermatozoa, led Zeller and 
Joel (1941) to suggest that the oxygen consumption in human semen 
is mediated chiefly by the spermine-diamine oxidase system. This 
requires further experimental proof. 

State of spermine in semen 

An interesting but as yet unsolved problem relates to the state 
of spermine in freshly voided semen. Some investigators envisaged 
the possibility that spermine occurs already in fresh semen as a 
phosphate salt which being poorly soluble, separates from the semen 
in the characteristic shape of Boettcher's crystals. However, in 
freshly ejaculated human semen there is not enough inorganic 
phosphate to combine with all the spermine and the content of 
inorganic phosphate increases on standing owing to the breakdown 
of phosphorylcholine (see p. 170). Furthermore, it has been the 
experience of all those who tried to obtain crystalline spermine 
phosphate from semen, that a successful crystallization can best be 
achieved with semen which has been allowed to stand for at least a 
few hours after ejaculation or by following Fuerbringer's recom- 
mendation and treating it with additional phosphate. Fuerbringer's 
(1881) interpretation of his own findings was that the basic com- 
ponent of Boettcher's crystals in ejaculated semen originates in the 

1 68 The Biochemistry of Semen 

prostatic secretion, whereas the phosphoric acid is derived from 
some other source. Recent advances in this field favour this hypo- 
thesis and indicate that the formation of spermine phosphate takes 
place only after the ejaculation, as the outcome of a reaction be- 
tween spermine which is contributed by the prostatic secretion, and 
phosphoric acid, which accumulates gradually through the action 
of the seminal phosphatases upon phosphorylcholine and perhaps 
also upon some other organic phosphorus compounds. 


The Florence reaction in semen 

Florence, working in the laboratory of forensic medicine in Lyons, 
made the following observation in 1895; if material stained with 
] luman semen is extracted with water, and a drop of this extract is 
mixed on a microscopic slide with a strong solution of iodine in 
potassium iodide (2-54 g. I2, 1-65 g. KI, 30 ml. water), the micro- 
scopic field is quickly filled with a mass of brown crystals which 
]-esemble closely Teichmann's crystals of haemin. Florence's treatise 
'Du sperme et des taches de sperme en medecine legal' (1895/96) 
created much interest in forensic medicine and led promptly to the 
lecognition of his test as a useful means for the identification of 
seminal stains. At first, a hypothetical substrate called 'virispermine' 
was held responsible for the formation of 'iodospermine' in the 
Florence reaction, but later on other substances came under investi- 
gation, including choline. All doubts concerning the nature of the 
Florence's reaction product were finally dispelled when Bocarius 
(1902) succeeded in converting 'iodospermine' preparations ob- 
tained from human and stallion semen, into a crystalline platinum 
compound which contained 31-62% Pt and was identical in every 
way with pure choline platinum chloride (31-64% Pt). Stanek's 
work in Prague (1905, 1906) had shown that the iodine compound 
formed in Florence's reaction was a water-insoluble periodide of the 
composition of an enneaiodide, corresponding to the formula 
QHuNOFIg. The method developed by Stanek for the quantitative 
determination of choline depended on the analysis of nitrogen 
(Kjeldahl) in the periodide precipitate; the more recent quantitative 

Spermine, Choline, Ergothioneine 169 

method of Roman (1930) is based on the same principle but involves 
an analysis of iodine instead of nitrogen 




Compared with other animal tissues and body fluids, semen ranks 
as one of the richest sources of choline. It owes its high choline 
content to the seminal plasma and not to spermatozoa as such. 
In rat, Fletcher, Best and Solandt (1935) found the following dis- 
tribution of total choline (mg./lOO g.): seminal fluid 514, brain 325, 
liver 260, pancreas 232, stomach 152, uterus 74, fat 23, blood 22. 
The composition is similar in other species, including man, where 
values exceeding 2000 mg./lOO ml. semen have been observed. This 
may explain a statement by Marcille (1931) that a positive Florence 
reaction can be obtained with dried human semen even when it is 
diluted with 1000 parts of water. However, there is no general 
agreement about the sensitivity of Florence's reaction. In fact, many 
investigators have criticized the reaction, mainly because the same 
specimen of semen will occasionally give a negative reaction at first, 
and a positive result later. This peculiar behaviour of human semen 
was elucidated by Kahane and Levy (1936, 1937) who discovered 
that human semen examined immediately after ejaculation contains 
practically no free choline, but that choline accumulates in semen 
gradually on standing, as illustrated by the following experiment: 
from 3-5 ml. semen mixed with 20 ml. water, consecutive 2 ml. 
samples were withdrawn and deproteinized by boiling with 9 ml. 
ethanol for 2 minutes; the quantitatively collected filtrates were 
evaporated, the residues extracted with dry ether and redissolved in 
water; choline was precipitated from the aqueous extracts with the 
Reinecke reagent and determined bromometrically. Results, which 

Table 23. Choline in human semen (Kahane and Levy, 1937) 

Time after 

ejaculation 2 min. 10 min. 1 hr. 6 hr. 22 hr. 48 hr. 120 hr. 
Choline liberated 

(mg. per 100 

ml. semen) 70 860 1600 2120 2030 2500 530 

170 The Biochemistry of Semen 

are given in Table 23, show a sharp increase in the choline content 
of human semen during the first hour of incubation, and the rela- 
tively slow accumulation during the next 47 hours; the terminal 
decline is probably due to bacterial contamination. 

Enzymic liberation of choline froin precursors in semen 

Following up their observation that choline accumulates in semen 
only after the ejaculation, Kahane and Levy demonstrated the pre- 
sence in fresh semen of a 'precurseur de la choline' which yields free 
choline as a result of hydrolysis which takes place in semen on 
standing. Apart from the seminal plasma itself, they found the 
choline precursor in various reproductive organs, including the 
testis of bull, boar, ram, stallion, rabbit and guinea-pig, the seminal 
vesicle of stallion and guinea-pig, and the epididymis of boar and 
ram, but not the prostate of dog, stallion or ram. However, the 
prostate, particularly that of dog, was found to be rich in the enzyme 
which splits off choline from the precursor. In a series of studies, 
Kahane and Levy (1938, 1945, 1949) have shown that the precursor 
is a water-soluble compound ('choline hydrosoluble combinee') 
which behaves like glycerylphosphorylcholine, and yields on incu- 
bation with prostatic extracts a mixture of free choline and in- 
organic phosphate; the quantity, however, of liberated choline was 
found to be far in excess of the simultaneously appearing inorganic 

Phosphorylcholine and glycerylphosphorylcholine 

The nature of the phosphorus compounds in semen which yield 
choline after ejaculation, was investigated by Lundquist (1946, 
1947(7, b) and by Diament, Kahane and Levy (1952). 

In human semen deproteinized freshly with trichloroacetic acid, 
the Danish investigator found 110 mg. acid-soluble P/100 ml, 
including 10 mg./lOO ml. of inorganic phosphate. On neutraliza- 
tion with barium hydroxide and precipitation with 2 vol. of ethanol, 
he recovered 60-70% of the phosphorus in the filtrate and from this 
he obtained by precipitation with mercuric chloride a fraction 
containing nitrogen and phosphorus in a ratio of approximately 1:1. 
The phosphorus compound thus separated was found to be very 

Spermine, Choline, Ergothioneine 171 

resistant to acid hydrolysis and no choline was set free from it after 
an hour's hydrolysis with N-H2SO4 (100^), long enough for glyceryl- 
phosphorylcholine, to release all its choline in a free form. On the 
other hand, under the influence of the prostatic secretion the com- 
pound yielded equivalent amounts of choline and inorganic phos- 
phate. All these facts pointed to the identity of the compound with 
phosphorylcholine, a substance previously isolated from beef liver 
(Inukai and Nakahara, 1935). Lundquist sought to obtain proof by 
preparing the calcium salt; this he found to be identical with the 
calcium salt of pure phosphorylcholine, C5Hi304NPClCa-4H20, 
obtained synthetically by the method of Plimmer and Burch (1937). 


HO— P— O -CHoCHa-N^ 

I " /|\ 

O ChJ CHp 



The distribution of phosphorylcholine in the human reproductive 
organs has not been investigated in detail, but Huggins and Johnson 
(1933) have good evidence that the bulk of the phosphorus present 
in the human seminal plasma is derived from the vesicular secretion. 
From this Lundquist infers that phosphorylcholine is formed in the 
seminal vesicles, and that the dephosphorylation is initiated at 
ejaculation, as a result of contact between the prostatic secretion 
which contributes the 'acid' phosphatase, and the vesicular secretion 
which provides the substrate; the optimum pH for the dephos- 
phorylation of phosphorylcholine by the prostatic phosphatase 
measured in acetate buffer solutions is about 6-3 (Lundquist, 1947a, 
b). It is of some interest to recall here the claim put forward by 
Kutscher and Sieg (1950) that preparations of both the 'acid' and 
the 'alkaline' phosphatases contain pyrophosphorylcholine as a 
characteristic constituent. However, Roche and his colleagues (1952) 
were unable to detect any cophosphatase activity in pure, synthe- 
tically prepared pyrophosphorylcholine. 

The possibility that compounds other than phosphorylcholine 
may act as precursors of free choline in semen was indicated already 
by the earlier observation of Kahane and Levy that the quantity of 

1 72 The Biochemistry of Semen 

choline liberated after ejaculation exceeds considerably the simul- 
taneously formed inorganic phosphate. Following up this observa- 
tion, the French investigators accomplished in 1952 the isolation of 
a second natural precursor of choline, namely glycerylphosphoryl- 
choline, from the seminal vesicle secretion of rats; the isolation and 
identification was performed as a ferric chloride compound (Dia- 
ment, Kahane and Levy, 1952, 1953). A similar result was obtained 
by Lundquist (1953) from his studies on the secretions of the seminal 
vesicles in rat and guinea-pig and the glandula vesicularis of rabbit. 

H,C— OH 


HgC— O— P— O— CH2 CH2N+ 




Physiological function of free and bound choline 

The occurrence of choline, phosphorylcholine and glycerylphos- 
phorylcholine in semen and in the accessory secretions naturally 
raises the problem of their physiological function. One possibility 
which merits serious attention, is that these compounds may be 
bound up specifically with the metabolism of phospholipids in 
either the male accessory organs or in the spermatozoa. 

The general importance of choline in the lipid metabolism of 
animals was first brought to light in 1932 when Best and his co- 
workers demonstrated that the appearance of the 'fatty livers' in 
rats fed a choline-deficient, high-fat diet, could be prevented by 
dietary supplements of choline. Researches which followed estab- 
lished two principal functions of choline, the lipotropic activity and 
the stimulating action on the turnover of phospholipids. In 1939, du 
Vigneaud and his co-workers discovered that choline is an important 
dietary source of methyl groups for the living animal, and this led 
to the recognition of choline as a participant in transmethylation 
processes. These three fundamental functions probably represent 
the clue to the understanding of the manifold symptoms associ- 
ated with choline deficiency. Among the various manifestations of 

Spermine, Choline, Ergothioneine 173 

choline deficiency those concerned with reproduction are particu- 
larly striking; choline is known, for example, to be essential for egg 
production in the chicken, as well as for normal lactation and 
nutrition in rats. 

The role of choline in transmethylations is linked with the 
presence of the trimethyl quaternary nitrogen. It is worth noting, how- 
ever, that while the phenomenon of transmethylation is common 
to a whole group of compounds bearing labile methyl groups, 
the lipotropic activity is restricted to choline and a few closely 
related derivatives. One of the lipotropically active derivatives is 
phosphorylcholine (Welch and Welch, 1938), and there is some 
evidence that the incorporation of choline into phospholipids pro- 
ceeds via phosphorylcholine (Wittenberg and Kornberg, 1953). 

A further possibility regarding the function of choline in semen 
comes to mind; choline and its derivatives belong to a group of sub- 
stances endowed with well-defined pharmacological properties, and 
it is not improbable that the base itself or one of its compounds may 
exert some pharmacodynamic effects either on the spermatozoa or, 
perhaps, on some parts of the male or female reproductive tract. 
When assayed by Goldblatt (1935^) on the m. rectus abdominis of 
the frog, 1 ml. human seminal plasma exhibited roughly the same 
activity as l^g. acetylcholine. There is, however, no chemical evi- 
dence to show that the substance in seminal plasma, responsible 
for this activity is in fact, acetylcholine. 

Choline esterase 

It has been claimed that sperm motility is somewhat increased 
by acetylcholine, and depressed by eserine, but this effect has never 
been analysed quantitatively and requires confirmation. There is, 
on the other hand, sufficient evidence to show that semen contains 
choline esterase as a normal constituent. In human semen, the con- 
centration of choline esterase was found to be low. Zeller and Joel 
(1941) using the manometric method, and employing a rather high 
concentration of acetylcholine as substrate, found that the quantity 
of acetic acid liberated by 1 ml. semen in 1 hr. is equivalent to not 
more than 70 [A. CO2, as compared with 3600 /td. in blood serum 
and 38000 /tl. in brain; moreover, the bulk of activity was derived 
from the seminal plasma and not from the spermatozoa. Boar 

1 74 The Biochemistry of Semen 

semen, on the other hand, has been found by Sekine (1951) to be 
highly active, the activity being more concentrated in the sperma- 
tozoa than in the seminal plasma. According to this author, boar 
spermatozoa, both epididymal and ejaculated, possessed choline 
esterase activity as high as that of brain, whereas the seminal plasma 
was only one-third as active as human blood serum. Boar sperma- 
tozoa, although highly active against acetylcholine, were found at 
the same time to be completely inactive against benzoylcholine 
which suggests that their choline esterase is of the 'true' or 'specific' 
type. Results obtained on ram semen (Legge and Mann, unpublished 
data) lead to a similar conclusion; ram spermatozoa exhibited a high 
activity at low concentrations of acetylcholine but were poorly 
active at high substrate concentrations, and hydrolysed efficiently 
acetyl-i9-methylcholine but were ineffective against benzoylcholine. 
A study was also made at the same time of the distribution of the 
enzyme between the sperm-heads and -tails, using ram spermatozoa 
disintegrated with glass beads in the Mickle mechanical shaker (see 
p. 87). Choline esterase occurred mainly in the tail fraction. 


Ergothioneine was first discovered by Tanret (1909) who isolated 
it from rye ergot. Two years later, Barger and Ewins (1911) identi- 
fied the new substance as a betaine of thiolhistidine (^-2-thiolglyoxa- 
line-4(5)-propiobetaine). The final confirmation of the structure was 
provided by Heath, Lawson and Rimington (1950, 1951) who suc- 
ceeded in synthesizing ergothioneine from 2-thiolhistidine; the latter 
is an amino acid which so far has never been found in nature 
but was prepared synthetically by Harington and his co-workers 
(Ashley and Harington, 1930; Harington and Overhoff, 1933), and 
shown by Neuberger and Webster (1946) to be unable to replace 
histidine as a growth-promoting factor in animals. 

/ \CH 

HS— C 


N^ +i(CH3)3 


Spermine, Choline, Ergothioneine 175 

Ergot from which ergothioneine has been obtained in yields 
varying from 65 to 260 mg./lOO g., remained the only natural source 
of this base until Hunter and Eagles (1925, 1927) isolated from pig 
blood a crystalline substance, named at first 'sympectothion', which 
gave with phosphotungstic and arsenophosphotungstic acid reagents 
the same blue colour as uric acid. Quite independently, a blood 
constituent with similar properties, named 'thiasine', was obtained 
by Benedict, Newton and Behre (1926). Somewhat later, both 
sympectothion and thiasine were shown to be identical with ergo- 
thioneine (Newton, Benedict and Dakin, 1926; Eagles and Johnson, 
1927). Blood ergothioneine, or 'thioneine' as it is sometimes called, 
occurs only in the erythrocytes and is not found in the plasma. In 
human blood there is no more than about 2 mg./lOO ml., but in the 
pig there may be as much as 26 mg./lOO ml. ergothioneine (Hunter, 
1951). Of the existing methods for the determination of ergothioneine 
that of Hunter (1928, 1949), based on the diazo reaction, is the most 
sensitive, specific and accurate. 

Isolation of ergothioneine from the boar seminal vesicle secretion 

It has been known for quite a while that protein-free extracts 
from semen exhibit a marked reducing power towards iodine, 
silver nitrate, 2 : 6-dichlorophenol-indophenol, and potassium per- 
manganate in the cold, and that this property is due to substances 
secreted in the seminal vesicle fluid. It has been mostly taken for 
granted however, that the reducing power of semen is due to ascorbic 
acid, particularly in the case of bovine and human semen (see p. 23) 
and no attempt was made to strengthen this assumption by a chemi- 
cal identification. In 1951, Leone and Mann undertook to purify 
the reducing substance from the boar seminal vesicle secretion, 
which being available in relatively large quantities, appeared to 
offer a convenient source of starting material. It was noticed in the 
course of the purification procedure that the reducing power went 
parallel with three other chemical properties of the boar vesicular 
secretion, (i) ability to reduce phosphotungstic acid to a blue 
reaction product, (ii) a strongly positive diazo reaction, and (iii) 
the occurrence of organically-bound sulphur which, however, unlike 
that present in glutathione, cysteine or methionine, could be oxidized 
and readily split off" as inorganic sulphate, by the addition of mild 

176 The Biochemistry of Semen 

oxidizing agents such as ferric chloride or bromine water. These 
facts suggested that the reducing substance under investigation may 
be the imidazole base ergothioneine. Further purification led to the 
isolation from 1300 ml. of boar vesicular secretion of 0-48 g. crystal- 
line material which was finally identified by analysis of sulphur 
(140%), nitrogen (18-3%), carbon (471%) and hydrogen (6-6%), 
and by other chemical means, as pure ergothioneine, C9H15N3O2S. 
With the isolation of ergothioneine from the boar vesicular secre- 
tion and boar semen, a rather unsuspected and abundant source 
of this sulphur-containing base in nature has been discovered. Un- 
like in blood, however, ergothioneine in the vesicular secretion is an 
extracellular constituent. Moreover, the concentration of ergothio- 
neine in this accessory secretion is much higher than in blood. In 
samples from twenty boars of the Large White and Essex variety, 
we found from 29 to 256 mg./lOO ml.; the average was 79 mg./lOO 
ml.; in boar semen itself the concentration is about 15 to 20 mg./lOO 
ml., but pig urine (boar and sow), and the foetal fluids contain 
practically no ergothioneine (Mann and Leone, 1953). 

The function of seminal ergothioneine and its behaviour towards 
sulphydryl-binding substances 

If ergothioneine possesses a specific physiological role in boar 
semen, this may well be linked, through its reducing sulphydryl 
groups, with a protective influence on spermatozoa. Boar semen, 
it must be remembered, differs from that of most other domestic 
animals by its exceptionally large volume and, at the same time, 
very low concentration of spermatozoa. Moreover, the period of 
time required for the completion of ejaculation is much longer in 
the boar than in other animals. Under storage conditions in vitro, 
the survival period of ejaculated boar spermatozoa compares on 
the whole unfavourably with that of ram and bull sperm. 

The results of investigations by Brachet (1944) and MacLeod 
(1951) have brought into prominence the importance of reduced 
sulphydryl groups for sperm motility, and, as previously mentioned 
(p. 58), substances with sulphydryl groups in a reduced form, such 
as cysteine or reduced glutathione, protect spermatozoa in vitro 
from the inhibitory action of SH-binding reagents. It is probable 
that glutathione plays actually a role in vivo since it has been shown 

Spermine, Choline, Ergothioneine 111 

to occur normally in spermatozoa (Infantellina, 1945; Tesoriere 
and Infantellina, 1946). Our researches (Mann and Leone, 1953) 
demonstrated that ergothioneine, which is a natural constituent of 
the seminal plasma, can counteract most efficiently the sperm- 
paralysing action of various thiol-reagents, including not only the 
mercaptide-forming and alkylating reagents but also substances such 
as o-iodosobenzoate which act by oxidizing compounds with SH- 
groups to the corresponding S-S derivatives. In fact, we were able 
to demonstrate the mutually antagonistic action of ergothioneine 
and o-iodosobenzoic acid in experiments with boar sperm taken 

60 90 

Incubation (min.) 
Fig. 16. Effect of ergothioneine on boar spermatozoa; anaerobic fructolysis 
at 37° in boar epididymal spermatozoa to which fructose was added, 

2-5 mg. fructose/10^ sperm; O O, no additions; x x, 

iodosobenzoate (IO^^m); • •, iodosobenzoate (10-^m) + 

ergothioneine (2 x 10" ^m). 

(Mann & Leone, 1953) 

178 The Biochemistry of Semen 

directly from the epididymis, in which unlike in the seminal vesicle, 
ergothioneine is absent. The epididymal spermatozoa were diluted 
with Ringer-phosphate-fructose, and the suspension divided in 
three equal portions; in one, serving as a control, fructolysis was 
measured directly, in another the reaction was allowed to proceed 
in the presence of lO^^^.iodosobenzoate, and in the third after the 
addition of the same amount of inhibitor, but together with ergo- 
thioneine, the latter in a concentration of the same order of magni- 
tude as actually found in vivo in the boar vesicular secretion. It 
can be seen from Fig. 16, that whereas the presence of iodoso- 
benzoate alone checked the process of fructolysis, the inhibition was 
prevented by the simultaneous addition of ergothioneine so that 
in effect, the spermatozoa were able to proceed with the normal 
utilization of fructose. 

Biogenesis of ergothioneine 

The mechanism of ergothioneine formation in the boar was 
studied by pursuing the fate of certain orally administered com- 
pounds labelled with radioactive sulphur, 253 (Heath, Rimington, 
Glover, Mann and Leone, 1953). It was found that inorganic sul- 
phate or thiolhistidine failed to provide a source of sulphur for 
ergothioneine in the boar; in this respect, the behaviour of thiol- 
histidine is of particular interest, since it demonstrates again that 
physiologically occurring substances need not necessarily arise from 
compounds to which they bear a close, though purely structural, 
chemical resemblance. Methionine, the amino acid pivotal in bio- 
logical transmethylations, was capable of supplying the sulphur for 
the biosynthesis of seminal ergothioneine. The spermatozoa them- 
selves also incorporated sulphur from labelled methionine but here, 
the maximum radioactivity appeared several weeks later than in 
the seminal plasma; this time-lag is presumably due to the fact that 
the processes of spermatogenesis, sperm maturation, and transport 
through the epididymis, require substantially more time than is 
needed for the formation and secretion of seminal plasma in the 
accessory organs. By administering to a living animal a labelled 
compound like methionine one might be actually able to deter- 
mine the time interval required for the processes of sperm formation 
and transport in the male reproductive organs. When synthetic 

Creatine and Creatinine 179 

35S-labelled ergothioneine was fed to the boar, some of it was ex- 
creted, unchanged, in the semen. This provides interesting evidence 
of the passage into semen of a substance absorbed from the ali- 
mentary tract. 

Since ordinary fodder contains no ergothioneine, there remained 
the possibiUty of its microbial formation in the digestive tract. 
This, however, was not borne out by an experiment in which 
aureomycin was fed to a boar, 1 g. daily for 24 days, but did not 
affect in any way the level of ergothioneine in semen. 

Human semen, and that of certain other mammals so far inves- 
tigated, was found to contain only a trace or no ergothioneine. In 
the bull, ram, and in man, the considerable reducing power of the 
seminal plasma towards dichlorophenol-indophenol is derived partly 
from ascorbic acid, but partly also from other reducing substances 
which await proper identification (Mann and Leone, 1953). An 
interesting approach in this direction was made by Larson and 
SaHsbury (1952, 1953) who reported on the presence in bull semen 
of an as yet unidentified reducing substance characterized by a 
positive reaction with sodium nitroprusside, and of sulphite. 


Occurrence in mammalian semen, and in the sperm and gonads of 

One of the earliest references to the presence of creatine and 
creatinine in male reproductive organs is to be found in a paper by 
Treskin who in Hoppe-Seyler's laboratory in 1872, isolated 016 g. 
pure creatinine from two pairs of bull testes. In 1923, Steudel and 
Suzuki isolated large quantities of crystalline creatinine, together 
with another nitrogenous base, namely agmatine, from ripe, fresh 
testicles of herring. Ilyasov (1933), using the colorimetric method 

/NH. / \ 


I " I 

CH3 CH3 

Creatine Creatinine 


1 80 The Biochemistry of Semen 

based on Jaffe's reaction, determined the creatine and creatinine 
content in bull and stallion semen. The mean values which he 
reported for creatine and creatinine were (mg./lOO ml.), 3 and 12-1 
in the bull, and 6-2 and 3-7 in the stallion, respectively. In the boar, 
the apparent creatinine content has been stated to be in blood 
plasma 2-4, in whole semen 0-3, and in the seminal vesicle secretion 
5-3 mg./lOO ml. (McKenzie et al., 1938). 

The generally held belief that in invertebrate animals arginine 
occurs in place of creatine is not supported by results of chemical 
analyses of gonads and sperm. Greenwald (1946) found in the 
sperm-laden nephridia of Echiurus 144 mg./lOO g. of apparent crea- 
tine, and 189 and 270 mg./lOO g. in the testes of Arbacia and 
Strongylocentrotus, respectively. He succeeded in preparing sub- 
stantial quantities of pure creatine and creatinine, from the testes 
and sperm of several invertebrates, including the sea-urchin (Stron- 
gylocentrotus), Urechis caupo, Holothwia tubiilosa, and Cuciimaria 
frondosa. In the case of the gonads of two ascidia, Microcosmus 
sulcatus and Boltenia, which contain chromogenic material, no 
creatine or creatinine could be isolated but 019 g. of pure betaine 
picrate was obtained from 150 g. of mixed gonads of Boltenia, 
indicating a concentration of at least 44 mg. of betaine per 100 g. 
of tissue. In the testes of arthropods, molluscs, and of a nematode 
(Ascaris), the amount of chromogenic material was so low as to 
indicate absence of creatine. 

Phosphocreatine and phosphoarginine 

The possibility that spermatozoa may contain creatine in the 
form of phosphocreatine was envisaged by Eggleton and Eggleton 
(1929) who found that the testes contain, next to skeletal muscle, 
the second largest concentration of phosphagen. Soon after the dis- 
covery by Parnas, Ostern and Mann (1934o, b) that extracts from 
skeletal muscles can synthesize phosphocreatine from creatine and 
phosphopyruvic acid, the same enzymic reaction was investigated 
in bovine epididymal spermatozoa by Torres (1935) who claimed 
that bull spermatozoa are definitely capable of such a synthesis. 
Her claim, however, has been refuted by Ivanov (1937) who failed 
to detect any synthesis of phosphocreatine in sperm, although he 
experienced no difficulty in confirming our results on muscles. 

Adrenaline and Noradrenaline 181 

More recently, Wajzer and Brochart (1947) reported on the isola- 
tion from boar sperm of a barium-precipitable fraction containing a 
mixture of two phosphagens, phosphocreatine and phosphoarginine. 
The distribution of the two phosphagens in the gonads and in 
sperm remains open to further investigations. An important contri- 
bution in this field was made by Green wald (1946) who isolated 
phosphocreatine in the form of a calcium salt, from the testes of the 


Occurrence in semen and accessory organs 

Using 66% ethanol for the extraction of various tissues v. Euler 
found that a substance closely resembling adrenaline is present in a 
particularly high concentration in the prostate gland of man, dog, 
rabbit and guinea-pig, in the seminal vesicle of bull and ram, and 
in the ampulla ductus deferentis of dog, bull and ram; the amount 
of the active substance corresponded to 1-5 //g. of adrenaline per 
g. of fresh tissue. 

Results obtained by Brochart (1948«) with the colorimetric method 
strengthened the view that adrenaline occurs as a normal consti- 
tuent in the semen of bull (1 /<g./ml.), goat (l-5-l-7/ig./ml.) and man 
(10-21 /ig./ml.); but later, Beauvallet and Brochart (1949) came to 
the conclusion that in the bull at any rate, the pressor activity of 
semen is due partly to adrenaline, and partly to noradrenaline. 
H H 

I I 




I I 

H H 

Adrenaline (epinephrine) Noradrenaline 

Enzymic oxidation 

When adrenaline or noradrenaline are added to bull semen in 
relatively high concentrations (10-100 /^g./ml.), the aerobic but 

182 The Biochemistry of Semen 

not the anaerobic, fructolysis is gradually inhibited. Brochart (1951) 
attributed this effect to adrenochrome and noradrenochrome which 
are formed aerobically through the catalytic action of the cyto- 
chrome system of bull spermatozoa upon adrenaline and noradren- 
aline, respectively. Adrenochrome itself, added to bull semen in 
amounts of 01-100 ^ag./ml, produces an instantaneous inhibition 
of lactic acid formation but only so long as it remains in the oxidized 
form; in the course of incubation with semen it becomes gradually 
reduced and inactive. 

However, an alternative mechanism for the oxidation of seminal 
adrenaline may well exist since Zeller and Joel (1941) have found 
in extracts from the human prostate and seminal vesicle, but not in 
the seminal plasma, a highly active monoamine oxidase (adrenaline 
oxidase). The reaction catalysed by this enzyme follows the equation 

and leads to the formation of hydrogen peroxide as one of the 
reaction products. 

Pharmacodynamic properties 

An interesting synergistic relationship between adrenaline and 
seminal plasma was reported by Goldblatt (1935Z)) who used 
isolated seminal vesicles of the guinea-pig as his test object. He 
observed that When he added 0-5-1 ml. human seminal plasma to the 
medium (30 ml. oxygenated Tyrode solution) there was usually 
no response from the vesicles; if, however, a small amount of 
adrenaline was added first and a considerable interval of time 
allowed to elapse until the only activity of the vesicle was an occa- 
sional contraction, then the addition of the seminal plasma pro- 
voked a succession of strong contractions. Adrenaline in doses so 
small as to be entirely devoid of activity alone, nevertheless induced 
in the vesicles a condition in which the seminal plasma itself or 
material obtained from it by ethanolic or acetone extraction, were 
able to develop to the full their pharmacological activity. This 
behaviour of adrenaline led Goldblatt to suggest that there may be 
a sort of synergism between adrenaline and the seminal plasma. 
But it is not clear as yet, whether effects of this kind are significantly 
related to the function of either the male or the female reproductive 


Citric Acid and Inositol 

Citric acid. Occurrence and distribution. Influence of male sex hormone. 
Citric acid in the female prostate. Metabolism and role of seminal citric 

Inositol. Occurrence and distribution. mesolnosiioX as a major con- 
stituent of the seminal vesicle secretion in the boar. Physiological function. 
Relation to other seminal constituents. 

Citric acid and inositol which will be considered jointly in this 
chapter, are both macro-constituents of the seminal plasma. In the 
past these two chemical substances, much like fructose, have re- 
ceived attention chiefly from plant biochemists, not unnaturally, 
since they occur in plants in much larger quantities and more 
commonly than in the animal kingdom. Similarly, ergothioneine was 
at first associated only with the fungi, until at a much later date 
small amounts of it have been detected in red blood cells and more 
recently, it was found to be a normal constituent of boar semen. 


Occurrence and distribution 

More than a century passed after Scheele's (1784) isolation of 
crystalline citric acid from lemon juice before this tricarboxylic acid 
was discovered in the animal body and identified as a major chemical 
constituent of milk, urine, bone and semen. The discovery in semen 
was made in Thunberg's laboratory at Lund, by Schersten (1929, 
1936), who noted that semen rapidly decolorizes methylene blue on 
addition of 'citrico-dehydrogenase', an enzyme prepared by Thun- 
berg from cucumber seeds. This observation was strengthened by 
chemical identification based on isolation of crystalline citric acid 
and the preparation of pentabromoacetone, a derivative formed 
from citric acid on oxidation with permanganate and bromine, in 


184 The Biochemistry of Semen 

a reaction described in 1897 by another Swedisli investigator, 





Citric acid 

Schersten enlarged his original finding by noting that citric acid 
in semen is derived from the male accessory organs of reproduction; 
in man, from the prostatic secretion, in the boar and bull, from the 
vesicular secretion. His findings have since been confirmed and 
extended by several investigators. In nine samples of human pros- 
tatic secretion Huggins and Neal (1942) recorded values ranging 
from 480 to 2688 mg. citric acid /1 00 ml., while two analyses of 
human seminal vesicle secretion gave 15 and 22 mg./lOO ml; in fif- 
teen specimens of human semen, the values ranged from 140 to 
637 mg./lOO ml. A survey by Harvey (1951), which covered 725 
specimens of human semen from 371 donors, revealed contents 
ranging from to 2340 mg./lOO ml.; the mean value of citric acid 
for the whole group was 479 mg./lOO ml. and 12-6 mg. /ejaculate. 
Citric acid also occurs normally in the semen of other mammalian 
species; a high concentration is characteristic for the bull (510- 
1100 mg./lOO ml.), boar (130 mg./lOO ml.), ram (110-260 mg./lOO 
ml.), and rabbit (110-550 mg./lOO ml.); rather lower concentrations 
are found in stallions (Humphrey and Mann, 1948, 1949). In some 
animals, e.g. the bull, ram, boar, and stallion, citric acid originates 
in the seminal vesicle, the same organ which also secretes fructose. 
In other species, however, the two substances are secreted in dif- 
ferent parts of the male reproductive system (Table 24). In the 
rabbit citric acid is limited largely to the gel-portion of semen, and it 
is produced by the glandula vesicularis, whereas fructose, it will be 
remembered, is secreted also in the prostate. An even clearer separa- 
tion occurs in the rat where fructose is found in the coagulating 
glands and in the dorso-lateral prostate, whereas citric acid is 
produced by the ventral prostate and the lateral lobes of the dorso- 
lateral prostate (Fig. 4). It is however, probable that even in species 
such as the bull, where citric acid and fructose are found side by 

Citric Acid and Inositol 185 

Table 24. Distribution of citric acid in male reproductive organs 
(Humphrey and Mann, 1949) 

Citric acid 

Species Material 

(mg./lOO g. fresh wt.) 

Boar: Secretion from Cowper's 




Epididymal semen 

Secretion from seminal 



Bull: Testis 




Secretion from the seminal 



Ampullar semen 


Epididymal semen 

Rabbit: Epididymis 




Glandula vesicularis 


Secretion of glandula 



Prostate (I, II and III) 


Cowper's gland 




Rat: Seminal vesicle proper 


Coagulating gland 


Dorsolateral prostate 


Ventral prostate 


side in the vesicular secretion, they may be secreted independently 
by different cells. A study of the bull seminal vesicle (Mann, Davies 
and Humphrey, 1949) has shown that the secretory epithelium is 
composed of three distinct types of cells, designated A, B and C, 
which appear to be concerned in the secretory processes, but react 
in a different manner to several histological stains. Type B con- 
sists of lipid-laden cells mentioned on a previous occasion (p. 133) 
whereas A and C cells contain no lipid, but still differ materially 
from each other, in so far as staining is concerned. It remains for 
future histochemical studies to establish their specific secretory 

186 The Biochemistry of Semen 

Influence of male sex hormone 

There is a close relationship between the formation of citric acid 
in the male accessory organs and the activity of the testicular 
hormone (Humphrey and Mann, 1948, 1949). Following castration, 
citric acid gradually disappears from the accessory gland secretions 
but reappears on implantation or injection of testosterone. In this 
respect, it behaves like seminal fructose, except, however, that in 
some animals (e.g. rabbit) the postcastrate disappearance and the 
hormone-induced reappearance of citric acid in the seminal plasma 
is not as prompt as that of fructose. The 'citric acid test' which 
depends on the relationship between the formation of citric acid and 
androgenic activity, has been successfully used in conjunction with 
the 'fructose test', for the study of certain endocrinological prob- 
lems, such as the time relationship between spermatogenesis and 
the onset of secretory function in male accessory organs (Mann, 
Lutwak-Mann and Price, 1948; Mann, Davies and Humphrey, 
1949; Mann, 1954); formation of citric acid in subcutaneous trans- 
plants from accessory gland tissues (Lutwak-Mann, Mann and 
Price, 1949); and determination of androgenic activity in ovarian 
hormones (Price, Mann and Lutwak-Mann, 1949, 1954). 

In castrated rats, a direct relationship exists between the dose of 
injected testosterone and the response of the seminal vesicle to 
produce citric acid (Mann and Parsons, 1950). This makes it pos- 
sible to utilize the determination of citric acid, like that of fruc- 
tose, as a sensitive and quantitative assay of androgen (Mann and 
Parsons, 1950). Removal of the hypophysis produces the same end- 
result as castration and again, the secretion of citric acid by the 
glandula vesicularis of a hypophysectomized rabbit can be restored, 
in this case, either by testosterone or by gonadotrophin (Mann and 
Parsons, 1950). The 'citric acid test' was also applied in studies 
concerned with the influence of malnutrition on the composition 
of semen (Lutwak-Mann and Mann, 1950^^, b, 1951; Mann and 
Walton, 1953). The effect of malnutrition manifests itself in a pro- 
gressive decline of the citric acid level in semen and accessory gland 
secretion and is due to a state of so-called pseudo-hypophysectomy 
(see p. 148). 

Citric Acid and Inositol 187 

Citric acid in the female prostate 

A gland corresponding in structure to the male prostate gland 
develops occasionally in the female body. It has been described in 
women but most studies concerning the so-called female prostate 
have been done with rats (cf. Price, 1944; Huggins, 1945; Mann and 
Lutwak-Mann, 1951^). In the rat this organ is located in a position 
similar to that of the male ventral prostate which it also resembles 
histologically. Ordinarily the incidence of prostate gland in the 
female rat is very low but by inbreeding it is possible to increase it 
to 80% or more. With rats from such a colony, Price, Mann and 
Lutwak-Mann (1949) have shown that the analogy between the 
female prostate and the male ventral prostate extends to the chemical 
character of the secretion and that, like its male counterpart, the 
female prostate produces citric acid, but no fructose. Injections of 
testosterone brought about a rapid growth of the gland, and a sharp 
increase in the output of citric acid. In response to daily adminis- 
tration of 200 /ig. testosterone propionate continued for three weeks, 
the average weight of the female prostate rose from 2 mg. to 1 12 mg., 
and the average content of citric acid in the gland from 2 yt^g. to 
125 /*g.; in male rats of comparable age the average citric acid 
content of the ventral prostate was 121 /^g. per organ. 

Metabolism and role of seminal citric acid 

It is still largely a matter of conjecture how citric acid is formed 
in the accessory organs. The rat seminal vesicle, which is a citric 
acid-producing organ is at the same time remarkable for its low 
content of aconitase (Humphrey and Mann, 1949), and on this 
ground one may be inclined to assume that perhaps citric acid 
accumulates because its further breakdown is prevented by the 
absence of this enzyme. On the other hand, however, the human 
prostate, which is also a citric acid-producing organ, has been found 
to contain aconitase (Barron and Huggins, \9A6b). A circumstance 
which may bear some relation to the mechanism of citric acid accu- 
mulation in the bull seminal vesicle, concerns the presence in this 
gland, and in its secretion, of a heat-labile factor which inhibits 
the enzymic breakdown of citrate by liver tissue (Humphrey and 
Mann, 1949). Yet another fact, mentioned briefly in conjunction 

188 The Biochemistry of Semen 

with the general chemical properties of accessory gland secretions 
(p. 19) concerns the high transaminase activity in the human pros- 
tate as well as in the rat ventral prostate, both of which secrete 
citric acid. The considerable transaminase activity together with the 
occurrence of free amino acids, including glutamic acid, in these 
two glands, point to the possibility of citric acid being formed from 
oxaloacetic acid which arises from glutamic acid as a result of 
transamination (Barron and Huggins, 1946o; Awapara, 1952a, b\ 
Awapara and Scale, 1952). 

As to its physiological role in semen, the available evidence does 
not support the view that citric acid influences markedly the aerobic 
or anaerobic metabolism of spermatozoa (Humphrey and Mann, 
1949); thus the beneficial effect of citrate on sperm motility ob- 
served by Lardy and Phillips (1945) may be due to a cause other 
than direct utilization by sperm. It is conceivable that citric acid 
is connected with the coagulation and liquefaction of semen and 
with the calcium-binding capacity of seminal plasma. In this con- 
nection one may recall the finding of Huggins and Neal (1942) 
that citrate in human semen causes prolonged coagulation of mix- 
tures of blood and seminal plasma, and that this delay in clotting 
can be effectively counteracted by calcium ions. The function of 
citric acid as a binding substance for calcium has been envisaged 
both by Schersten (1936) and Huggins (1945) and it is certainly sig- 
nificant that milk and bones, both rich in citrate, also have a high 
calcium content. Perhaps in the absence of citric acid in the prostatic 
secretion, there would be an even higher incidence of calculi and 
stones. The possibility of a link with the hyaluronidase activity 
cannot be excluded, as indicated by Baumberger and Fried (1948) 
who found that citrate exerts a protective action against so-called 
antinvasin in vitro. Lundquist (1947Z?), however, believes that citrate 
may act as an activator of the prostatic 'acid' phosphatase. Lastly, 
let it be remembered that citric acid, in combination with potassium 
and sodium ions, may play a part in maintaining the osmotic 
equilibrium in semen. Our own studies on the boar vesicular secre- 
tion (Mann, 1954) point in this direction. 

Citric Acid and Inositol 189 


Occurrence and distribution 

Inositol was first discovered in 1850 by Scherer, at Wiirzburg, 
who isolated it from the mother-liquor remaining after the separa- 
tion of creatine from beef meat, as a crystalline, colourless and dis- 
tinctly sweet-tasting substance. He named it 'inosit' to underline its 
origin from muscle, and showed that its composition and properties, 
except for lack of reducing capacity are similar to those of a hexose. 
In 1887 Maquenne proved that this non-reducing and optically- 
inactive compound is not a sugar in the strict sense but a hexitol 
derived from cjc/ohexane. In the years which followed, Bouveault 
(1894) and others brought forward evidence for the existence of 
several cyclitols derived from cjc/ohexane; since then it became 
customary to define the compound from muscle as m^^oinositol, in 
distinction to the other isomers. The configuration of meso'mosiioX 
was finally established in 1942, by Dangschat in Fischer's laboratory, 
and by Theodore Posternak, in Switzerland. 

H H 

1 I 

C C 

H / 1 I \ OH 

1/ OH 0H\| 

c c 

I V OH H / I 

I I 



Apart from muscle, inositol has also been isolated from urine 
(Cloetta, 1856), and from green beans (Vohl, 1856). Later, several 
plants were found to yield on extraction such very large amounts 
of this cyclitol, that like citric acid, me^oinositol came to be 
regarded generally as a typical plant constituent, a view strengthened 
by the discovery of phytic acid (inositol hexaphosphate) in grain, 
and lipositol (a monophosphoinositol-containing phosphatide) in 
soya-bean. The interesting history of these and later developments 
in the biochemistry of inositol will be found in the monograph by 

190 The Biochemistry of Semen 

Fleury and Balatre (1947) and in the reports by Courtois, Fleury, 
Posternak, and Schopfer, forming part of a (1951) symposium. 

Inositol as a major constituent of the seminal vesicle secretion in the 

In 1951, in the course of investigations on ergothioneine, the 
author noticed that a large ethanol-precipitable fraction could be 
separated from the boar vesicular secretion, containing no ergo- 
thioneine, citric acid, or fructose, and almost free from sulphur, 
nitrogen, or phosphorus. On further purification, from 1 litre of 
the vesicular secretion, 18 g. of crystalline material was obtained 
which had a pronounced sweet taste, but was non-reducing and 
optically inactive. The substance had a m.p. of 225° which is that 
of pure m^'^cinositol, and contained 40-28 °o carbon and 6-79% 
hydrogen, as against 40-ll°o carbon and 6-66% hydrogen, theoreti- 
cally expected from inositol. The Scherer-Salkowski reaction per- 
formed with 01 mg. was strongly positive, and on oxidation with 
periodic acid the substance isolated from the seminal vesicle secretion 
showed a titration curve identical with that of pure mesomo?,\Xo\. 

This and subsequent experiments (Mann, 1951c, 1954) showed 
that the boar vesicular secretion is the richest source of free 
inositol in nature, and that between 40 and 70% of the dialysable 
contents of this biological fluid is made up of inositol. Table 25 
shows the results of chemical analyses of the vesicular secretion 
from five boars, carried out in each instance on fluids collected 
separately from the left and right gland. As can be seen, the 
inositol content of these fluids was 208 to 2-64%, the variations 
being much smaller than they would be in the case of fructose or 
citric acid. It is also of some interest to note that the left and the 
right seminal vesicle produced secretory fluids which were alike in 
quantitative composition. The average values (mg./lOO ml.) based 
on the analyses of five pairs of secretions were: fructose 65, citric 
acid 381, ergothioneine 91, inositol 2414. 

Inositol is restricted in its distribution to the seminal vesicle, and 
is not present, at any rate in appreciable quantities, in the secretions 
from the boar epididymis or Cowper's gland. Furthermore, it 
appears to be rather specific for the boar, so far as can be judged 
from preliminary experiments carried out with the semen of other 

Citric A cid and Inositol 191 

Table 25. Individual variations in the composition of boar vesicular 
secretion (Mann, 1954) 

(Analyses carried out separately on the fluids collected from the left 
(L) and right (R) seminal vesicle of five boars, nos. I-V.) 


Total volume of secretion 

from both vesicles (ml.) 






Total weight of the empty 

vesicles (g.) 






Concentration in the vesicu- 

lar secretion 

(mg./lOO ml.) 














Citric acid 














= (L) 

































(R) 2355 2610 2640 2345 2080 

Species. Bull semen is poor in inositol, and human semen, according 
to Nixon (1952), contains usually less than 01%. 

Physiological function 

In the past, the physiological function of inositol has been 
associated mainly with nutrition, particularly since Woolley's (1944) 
important observations on the curative effect of inositol in a 
dietary dystrophy in mice, coupled with retarded growth and 
alopecia. Inositol has also been known to remedy a certain type 
of fatty liver in rats (Gavin and McHenry, 1941; MacFarland and 
McHenry, 1948), and its role as a lipotropic factor has been stressed 
repeatedly. An observation, however, which merits particular atten- 
tion in view of its bearing on animal reproduction, concerns a 
peculiar disturbance in hamsters: if hamsters are raised on an 
inositol-deficient diet, they tend to produce dead litters or die in 
parturition (Hamilton and Hogan, 1944). Yet another aspect of the 

192 The Biochemistry of Semen 

function of inositol became apparent when Chargaff and his co- 
workers (1948) reported that in tissue cultures inositol protects 
dividing fibroblasts from the toxic effect of colchicine and other 
mitotic poisons. 

It remains for future studies to determine more fully the function 
of inositol in boar semen. Judging from the author's own experi- 
ments there is little evidence that inositol is metabolized directly by 
spermatozoa. One is inclined to assume for inositol a role in the 
maintenance of the osmotic equilibrium in boar seminal plasma, 
seeing that the seminal vesicle secretion, unlike other body fluids 
of the pig, is almost completely devoid of sodium chloride (Mann, 
1953, 1954). 

Relation to other seminal constituents 

The mechanism by which inositol is formed in mammals is 
obscure but two tentative hypotheses have been put forward in the 
past. One involves its formation from a derivative of phosphoinositol 
such as for instance, lipositol. Fischer (1945) however, believes that 
inositol acts in the animal body as a sort of chemical intermediary 
between the sugars and certain aromatic substances, or alternatively, 
as a reserve carbohydrate for hexoses. The close structural similarity 
of inositol to glucose as well as to fructose, is certainly a point 
which must be considered in future investigations on the origin 
of seminal inositol. 


A STRIKING feature of semen which did not escape Leeuwenhoek, 
and which has been abundantly and repeatedly confirmed since, is 
the extraordinary diversity of shape and structure, encountered 
among the spermatozoa of different species. It even led Wagner and 
Leuckart (1852) to state 'that one may often safely venture to infer 
from the specific shape of these elements the systematic position 
and the name of the animals investigated'. Similarly, anatomists and 
physiologists aUke, have long accepted as natural the existence of 
remarkable species variations in the form and size of the male 
accessory glands, the organs responsible for the elaboration of that 
apparently indispensable adjunct of spermatozoa, the seminal 
plasma. It behoves us, I feel, to adopt a similarly enlightened atti- 
tude of mind towards the chemistry of semen. Is it not rather un- 
reasonable to expect that chemical findings made with the semen of 
one species must needs extend to that of others? The fact that a 
given substance is found in substantial amounts in the semen of one 
species, but is missing in others, by no means detracts from its 
physiological value: on the contrary, it is highly probable that such 
species-restricted occurrence is intimately linked with some other 
biological characteristics, peculiar to certain, but not necessarily 
all, animal species. 

A critical approach, free from bias, is also called for in the 
comparative evaluation of the morphological and chemical findings 
in semen. To expect, as has been done, the existence of a strict cor- 
relation between say, the fructose level in seminal plasma and sperm 
density in semen, is no more justifiable than to look for a relation- 
ship between, for instance, the glucose level in blood plasma and 
the number of red cells in blood. Similarly, although the secretion 
of fructose in the accessory organs depends closely upon the activity 
of the male sex hormone, it would be mistaken to attribute the level 
of fructose in semen to the influence of this hormone alone, because 
in reality it is conditioned by a multitude of other factors, including 
the general nutritional state of the body, size and storage capacity 


194 The Biochemistry of Semen 

of the accessory glands, frequency of ejaculation, volume of semen» 
ratio between sperm and seminal plasma, and last but not least, the 
blood glucose level. Above all, it is essential to bear in mind that 
profound changes in the composition of semen, elicited in response 
to drastic experimental procedures like castration or hypophy- 
sectomy, are unlikely to be equalled in extent by those encountered 
in hormonally deficient humans or in large domestic animals. 

Having thus come to the end of my discourse, I would like to 
leave the last word with Leeuwenhoek; when reporting in 1677 to the 
Rt. Hon. the Viscount Brouncker, President of the Royal Society, 
upon the progress of his researches on semen, he felt it incum- 
bent upon him to add: .'If your Lordship should consider that these 
observations may disgust or scandalize the learned, I earnestly beg 
your Lordship to regard them as private and publish or destroy 
them, as your Lordship thinks fit.' 


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Abalone, egg-membrane lysin, 95 
Abnormalities in semen, 11, 39 
Absorption spectrum of cyto- 
chrome, 90; intensification by 
liquid air, 65, 91 
Accessory glands, structure, 15-16; 
secretory function, 15-29, 137- 
50, 167, 172, 176-7, 184-8, 
191-4; onset of activity, 140-1, 
186; action of secretions on 
sperm, 24, 55; dependence on 
androgens, 67, 139^6, 186-7; 
pharmacological effects, 25-7; 
after irradiation, 59-60; lipids, 
130-3; utilization of sugars, 150; 
fructose, 137-8; citric acid, 184-8; 
changes in malnutrition, 148, 186 
Acetal phospholipids, 126-7 
Acetic acid, formation, 51; as sub- 
strate, 52 
Acetylcholine, 27, 173 
Acidity, effect on sperm, 60-1 
Acid phosphatase, in semen and 
accessory secretions, 16-17, 118- 
20; during spermatogenesis, 5; in 
galea capitis, 13; histochemical 
detection, 13; dependence on 
androgens, 68, 118-20; purifica- 
tion, 119-20; role in choline 
formation, 119, 168; role in 
spermine phosphate formation, 
167-8; activation by citric acid, 
Acid-soluble phosphorus, 20-2, 

32-3, 36, 107, 128, 191 
Aconitase, 187 
Acrosome, 12; PAS-reaction, 12; 

indophenol reaction, 90 
Adenine, 100, 101, 116, 155 

Adenosine diphosphate, 156 
Adenosinetriphosphatases, 86, 122- 

3; and sperm activity, 14, 155-8 
Adenosine triphosphate, distribu- 
tion and function in semen, 107, 

129, 155-8; after irradiation, 59; 

during senescence, 77 
Adenylic acid, dephosphorylation, 

Adrenaline in semen and accessory 

organs, 27, 181-2 
Adrenaline oxidase, 182 
Adrenochrome, effect on sperm, 

Age of sperm {see also ripening), 

Agglutination of sperm, 70, 79, 114 
Agmatine, 179 
Alanine, in semen and reproductive 

organs, 86, 106, 113; in diluents, 

Albumin, in semen, 112; effect on 

sperm, 76 
Albumose in semen, 1 1 1 
Algae, carotenoids in reproduction, 

Alkaline phosphatase, 120-1, 150; 

during spermatogenesis, 5; role 

in fructose formation, 150-1 
Alkalinity, effect on sperm, 61 
Alkaloids, effect on sperm, 56; in 

chemotaxis, 71-2 
AUelostasis, 74 
AUomyces, carotenoids in gametes, 

Alloxan diabetes, effect on seminal 

fructose, 146 
Amino acid oxidase in sperm, 117 
Amino acids, free in semen and 

prostate, 19-20, 86, 111-13, 188; 

in seminal proteins, 84-7, 106-10; 



The Biochemistry of Semen 

Amino acids (contd.) — 

in diluents, 76, 114; effect on 
sperm, 76, 114; after castration, 

Aminopeptidase, 1 1 6 

Aminosugar, 21, 36, 112 

Ammonia, 21, 32-3; formation, 
111, 116-17 

Amphibia {see also frog), sperm- 
affecting agents, 54, 95; nucleic 
acid, 105 

Ampulla, secretory function, 15, 
24, 113, 138, 181, 185 

Androgamones, 70-1; 95-6 

Androgens, in semen, 67-9; in 
ovaries, 144; indicator tests based 
on semen analysis, 67-8, 118-19, 
139-45, 186-7; effect on acces- 
sory organs, 67-8 

Antibiotics, use in semen diluents, 

Antibodies in semen, 79-80 

Anti-fertility factor, 98 

Antifertilizin, 70 

Antifibrinolysin, 115 

Antigens in semen, 79-80 

Arbacia {see also sea-urchin), pH 
of semen, 44; sperm nucleic acid, 
104; creatine, 180; gelatin-liquefy- 
ing enzyme and mucopoly- 
saccharase, 96; echinochrome, 
70; protein, 84; effect of seminal 
plasma and amino acids on 
sperm, 76 

Arbacine, 108 

Archegonia, chemotropic sub- 
stances, 72 

Arginine, free, 113; protein-bound, 
84, 106-8 

Arniadillidium vulgare, sperm sur- 
vival in the female tract, 9 

Arsenicals, effect on sperm, 

Artificial diluents, 55, 73, 76, 79-82 

Artificial insemination, 3, 38-40, 

Arvelius albopunctatus, PAS-reac- 

tion in the acrosome, 11-12 
Ascaridine in Ascaris megaloce- 

phala, 85 
Ascidia, betaine in gonads, 1 80 
Ascorbic acid, 20, 23, 32-3, 175, 

Asellus aquaticus, spermatogenesis 

and ribonucleic acid, 5 
Ash content of semen, 32-3, 43, 87 
Aspartic acid, free, 86, 113; in 

proteins, 85 
Ass semen, 2, 43 
Aureomycin, in diluents, 81; effect 

on biosynthesis of ergothioneine, 

Axial filament, 13 
Azide, effect on sperm, 56, 91 
Azoospermia, due to hyperpyrexia, 

62; urinary excretion of steroids, 

62; fructose and fructolysis, 46, 


Bacteria in semen, 39, 81 

Bacteriostatic properties of sper- 
mine, 162 

Barberio reaction, 163^ 

Bat semen, volume, 2; sperm dens- 
ity, 2, 82; survival of spermato- 
zoa, 6, 9, 82; clotting, 28; 
dilution effect, 82 

Bee, clotting of semen, 28 

a-Benzoinoxime (cupron), as a 
metal-binding agent, 76 

Benzoquinone, effect on sperm, 57 

Betaine, in gonads of ascidia, 180 

Bicarbonate, in semen and acces- 
sory secretions, 18, 22, 32-3; in 
diluents, 81 

Blood glucose, as precursor of fruc- 
tose, 149 

Blood plasma and serum, effect on 
sperm, 55, 79 

Boar accessory organs, 16, 35-6 

Boar semen, volume and density, 2, 
35-6; dry weight, 32; physico- 



chemical properties, 41-4, 60; 
sperm structure, 1 1 ; gelation, 28, 
35; protracted period of ejacula- 
tion, and fractions, 28, 35-7; 
nitrogenous compounds, 32; cit- 
ric acid, 36, 184-8; creatinine, 
phosphocreatine and phospho- 
arginine, 180-1; ergothioneine, 
36-7, 175-9; fructose, 32, 36-7, 
136; lactic acid, 36; lipids, 32, 
126; mineral constituents, 32, 36; 
protein and proteose, 32, 113; 
choline esterase, 173-4; hyalu- 
ronidase, 94; respiration, 50, 60; 
resistance to dilution, 78; time 
required for spermatogenesis and 
ripening, 178-9 

Boar vesicular secretion, contribu- 
tion to semen, 35-7; composi- 
tion, 20-4, 35-7, 175-6, 185, 191; 
individual variations, 191; fruc- 
tose, 35-6, 191; inositol, 190-2; 
ergothioneine, 175-6; citric acid, 
184-8; creatinine, 180; osmotic 
equilibrium, 188, 192 

Boettcher crystals, 161 

Bombyx mori, sperm and accessory 
secretions, 25 

Bouchon vaginal, 28-9 

Bracken sperm, 72, 92 

Broad helix, see mitochondrial 

Buffering capacity of semen, 40, 45 

Bull ampullar secretion, adrena- 
line, 181; citric acid, 185; fruc- 
tose, 16 

Bull semen, volume and density, 2; 
dry weight, 32, 85; ash, 87; 
physico-chemical properties, 7, 
8, 41-5, 60; nucleic acid, 5, 105; 
metabolism and its relation to 
motility and fertihty, 45-53, 58, 
77, 128, 152-4; seminal fractions, 
38; time required for sperm to 
reach the ovum, 9; sperm struc- 
ture, 10-15; separation of sperm 

parts, 85-7; PAS-reaction, 12; 
spermatogenesis and vitamin A, 
72; malnutrition effects, 149; cas- 
tration and hormone effects, 
142-9; vasectomy effect, 113, 138; 
androgen content, 68-9; diluents, 
77-8, 80-2; composition, 32-5, 
84-7; proteins and proteose, 32, 
84-7, 110; sulphur, 85-7, 179; 
amino acids, 85-7, 113; amino 
acid oxidase, 117; phosphorus 
compounds, 32, 125-8, 155, 180; 
phosphatases, 120-3; hyaluroni- 
dase, 94; iron, 88, 109; haematin, 
88; catalase, 92; cytochrome, 90; 
nitrogenous bases, 180-2; uric 
acid, 32, 123; citric acid, 32-3, 
184-8; inositol, 191; lipids, 125- 
8; reducing substances, 23-4, 179; 
fructose and fructolysis, 32-5, 
46-8, 135-9, 152-4 
Bull seminal vesicles, secretory 
epithelium, 185; secretory func- 
tion 16, 20, 34, 38, 137; hormonal 
aspects, 137-42; role in fructose 
formation, 16, 137-8, 142, 149-51 
Bull vesicular secretion, composi- 
tion, 20^; lipid, 133, 185; adrena- 
line, 181; fructose, 138, 142; citric 
acid, 184-8; flavin, 22; reducing 
substances, 23-4; phosphatases, 
118-23; xanthine oxidase, 123 
Bursa copulatrix, role in insects, 9 
Butylquinone, effect on sperm, 57 
Butyric acid, as substrate, 52 

Calcium, 18, 21, 32-3, 36, 77, 132, 

Cap (sperm-cap), 12-13 

Capacitation, 8-9 

Carbohydrate {see also individual 
sugars), in gynogamone, 71; in 
semen, 21-4, 30-8, 135-7; meta- 
bolism, 45-53, 134-59 

Carbon dioxide, see bicarbonate 

Carbonic anhydrase, 19, 87, 89 


The Biochemistry of Semen 

Carbon monoxide, effect on sperm, 
73, 91-2; reaction with cyto- 
chrome oxidase, 91 

Carotenoids, associated with 
gametes and reproduction, 72-3 

Carp sperm, 104, 106, 181 

Castration effects, 113, 119, 139- 
45, 186 

Cat, 21; lipid bodies, 131 

Catalase, 92-3 

Centrifugation, effect on sperm, 83 

Centriole, 14 

Centrosome, 13 

Cephalin, 124, 130 

Cervical mucus, penetration test, 
40; as medium for sperm, 40, 54 

chloride, effect on sperm, 57 

Cetyltrimethylammonium bromide, 
(CTAB), spermicidal properties, 
50, 57; effect on succinate oxida- 
tion, 50 

Charcot-Leyden crystals, 161 

Chemotaxis, 71-2 

Chiroptera, copulatory plug, 28 

Chloride, 18,21,23,32-3, 191 

Chloroform, effect on sperm, 54 

Cholesterol, 86, 124-5, 130-2 

Choline, 119, 130, 168-74 

Choline esterase, 173^ 

Chromatin, in sperm nucleus, 11, 

Chromosin, 109 

Chromosomin, 109 

Citraconic acid and chemotaxis, 72 

Citric acid, in semen and reproduc- 
tive organs, 16-24, 31-3, 132, 
183-8, 191; relation to andro- 
gens, 68, 139, 186; metabolism 
and function, 187-8; association 
with gel, 31, 34, 184, 188; in 
diluents, 81; in prostatic calculi, 

Citric acid test, for androgens, 68, 

Clam, nucleoprotein, 100 

Clotting, of semen, 28-9, 114, 188 
Clotting test, for androgens, 67 
Clupeine, 106-8 
Coagulating gland in rat, 16, 29; 

fructose secretion, 16, 138^5; 

transplantation, 144 
Coagulation of semen, 28-9, 114, 

Cock semen {see also fowl), volume 

and density, 2; sperm structure, 

11; composition, 33; pH, 44, 60; 

respiration, 50; irradiation effect, 

59; diluents, 79-81; nucleic acid, 

105; lipid capsule, 126; sugar, 137 
Cod-fish, gadushistone, 108; lipids, 

Coelenterate, sperm nucleic acid, 

Cold effect on sperm, 62-6 
Colostrum corpuscles, 131-2 
Colour {see also pigments), 1, 17, 

Conception rate, and quality of 

semen, 39, 42 
Contraception {see also spermicidal 

substances), 55-8, 79-80, 97-8 
Contractive substances, in sperm, 

14; in seminal plasma, 27 
Copper, 88-9; toxicity, 54, 58, 76 
Copulatory plug, 28-9 
Corpora amylacea, 131-3 
Cortical helix, 15 
Cowper's gland, 15, 35, 185 
Cozymase, in sperm, 157-8; dephos- 

phorylation, 122 
Creatine, 179-81 
Creatinine, 179-81 
Crocetin, dimethyl esters, in Chlamy- 

domonas, 72 
Crocin, in Chlamydomonas, 72 
Crustaceans, nucleic acid, 104 
Cryptorchidism, 62 
Cumulus-dispersing factor, and 

hyaluronidase, 96 
Cyanide, effect on sperm, 55, 56, 91, 




Cyclop terin, 106 

Cyprinine, 106 

Cysteine, effect on sperm, 58, 154, 

Cystine, in semen, 85, 87, 110, 

Cytochrome, in sperm, 14, 66, 87, 
90-2; leakage from sperm, 10, 
57, 83; role in metabolism, 90-2, 

Cytochrome oxidase, 14, 87, 90-2 

Cytometer, 39 

Cytoplasm of sperm, 85-7; baso- 
philic character, 5; dehydration 
changes, 7 

Cytosine, 100-1 

Deer, sperm lipids, 6 

Dehydration changes in sperm, 7-8 

7-Dehydrocholesterol, 133 

Dehydrogenase activity, 48, 87, 92 

Dehydro/j'oandrosterone, 68-9 

Deoxyribonucleic acid, in sperm, 
87-8, 98-107; Feulgen reaction, 
11, 100-1 

Deoxyribonucleoproteins of sperm, 
5, 8, 11, 86, 98-100, 106-9 

Deoxyribose, 100 

Deproteinizing agents, 1 1 3 

Detergents, spermicidal properties, 
50, 57; mode of action, 57 

Diabetes, effect on seminal fruc- 
tose, 146-8 

Diamine oxidase, 167 

Diastase, 17 

Diazo reaction, 175 

Dichlorophenol indophenol, reduc- 
tion, 23, 175, 179 

Diethyldithiocarbamate, as metal- 
binding agent, 76 

Differential (live-dead) staining, 39, 

Diluents, 55, 73, 76, 79-82 

Dilution effect, on sperm, 54, 73-9 

2 : 4-Dinitrophenol, effect on sperm, 
52, 56 

1 : 6-Diphosphofructose, dephos- 
phorylation, 36, 118-21, 150; as 
intermediary in glycolysis, 38-9, 
150, 157 

Dirscherl-Zilliken reaction, 68 

Disintegration of sperm, 86-7, 

Dodecylsulphate, as spermicidal 
agent, 50, 57 

Dog ampullar secretion, adrenaline, 

Dog prostate, adrenahne, 181; 
choline-liberating enzyme, 170; 
free amino acids, 20; hyper- 
trophy, 20, 133; corpora amyl- 
acea, 131-3 

Dog prostatic secretion, composi- 
tion and properties, 17-18; 
enzymes, 17, 115; lipids and lipid 
bodies, 126, 131-3; effect on 
sperm, 25 

Dog semen, volume and density, 2 
physico-chemical properties, 28 
41-44; composition, 33; tempera 
ture effect on sperm, 62, 63 
ethanol effect on sperm and fer 
tihty, 56; resistance to dilution 
78; hyaluronidase, 94; fibrino 
genase, 28; acid phosphatase, 29 
lipids, 126, 131; fructose and 
citric acid, 33; respiration, 50: 
effect of sugars on motility, 151-2 

Dogfish, fructose, 136 

Dry weight, of semen, 21, 32-3, 36, 

Echinarachnius parma (ssind-doWar), 
cyanide effect on sperm, 92 

Echinochrome, 70 

Echinoderms, (see also sea-urchin 
and sea-cucumber), sperm nu- 
cleic acid, 104 

Echinus esculentiis {see also sea- 
urchin) semen, density, 74; pH 
44; dilution effect, 74; oxygen 
effect. 74; glycogen, 5; nucleic 


The Biochemistry of Semen 

Echinus esculentus (contd). — 
acid, 104; lipids and phospho- 
lipids, 125, 129; sterols, 125; 
fatty acids, 125; adenosine tri- 
phosphate, 155 

Echiurus, creatine, 180 

Egg-membrane lysin in molluscs, 95 

Egg-surface liquefying agent, and 
androgamone, 23, 71 

Egg-water, agglutinating and ac- 
tivating effects, 70-6, 114 

Egg-yolk, diluents, 80; eflfect on 
sperm respiration, 117; glucose 
content, 157 

Ejaculate, species characteristics, 2; 
fractions, 9, 28, 35-8; frequency, 

Ejaculatory duct, obstruction, 137- 

Electrical impedance changes, 41-2 

Electric stimulation of ejaculation, 

Electro-conductivity, 41 

Electrolytes, 18, 23, 25, 32-3; inter- 
relations, 42-4, 73-7, 188, 192 

Electron microscopy, application 
to sperm, 11, 14-15 

Electrophoresis of seminal pro- 
teins, 112 

End-piece, characteristics, 15 

Enolase, 158 

Enzymes, in seminal plasma, 15-29, 
114^23, 167, 170-3; in sperm, 5, 
13, 83-9, 117, 151-9, 173-4, 181- 
2; release from damaged sperm, 
10, 64, 83, 94-5; in metabolism, 
43-53, 128-30, 149-59, 187-8; 
aflfecting sperm-fibrils, 14 

Epididymal spermatozoa, 6-8, 24- 
5, 82, 86-8, 125, 129, 152-4, 

Epididymis, as storage organ for 
sperm, 6, 15,25, 35-7, 82, 1 54, 1 78, 
185; secretion, 25, 35-6; ripening 
effect, 25, 154; dependence on 
androgen, 67; oxygen tension, 61; 

citric acid, 185; dehydrocholes- 
terol, 133 

Epinephrine (adrenalme), 181-2 

Ergothioneine, in semen and acces- 
sory secretions, 15, 20-3, 36-7, 
175-8, 191; counteraction of 
thiol-reagents, 58, 176-8; role, 
176-8; biogenesis, 178 

Esocine, 106 

Esox lucius (pike), nucleic acid, 105 

Ethanol, effect on sperm, 56; 
excretion in semen, 56 

Ethylenediamine tetra-acetate, as 
metal-binding agent, 76 

Ethylquinone, effect on sperm, 57 

Evaluation of semen quaUty, 38-53, 
78, 92-3, 138 

Exhaustion effect, 34 

Extraneous factors, influence on 
sperm, 54-82 

Fatty acids, in semen, 125-7; 
utilization, 52 

Fatty aldehydes, in acetal phos- 
pholipids, 126 

Female prostate, 187 

Female reproductive tract, role in 
sperm transport, 8-9 

Fertility, evaluation through semen 
analysis, 3, 38-53, 78-82, 92-5, 
138; after irradiation, 58-60; 
temperature effects, 62-66; of 
epididymal sperm, 6-8; and 
artificial diluents, 79-82; relation 
to 'lytic' agents and hyaluroni- 
dase, 95-7; in malnutrition, 148- 
9, 172-3, 191; contraception and 
'anti-fertihty factor', 55-8, 79- 
80, 97-8 

Fertilization, 8-10, 69-73, 95-8 

Fertilizin, 70-1 

Feulgen reaction, 11, 100-1, 126-7 

Fibrils, in spermatozoa, 13-14 

Fibrin, seminal, 114 

Fibrinogenase, 17, 29, 114-15 

Fibrinolysin, 17, 29, 114-15 



Fish semen {see also individual 
species), pH and dilution effects, 
60, 74-5; proteins, 85-6, 106-9; 
amino acids, 113; lipids, 124-5; 
cholesterol, 124; nucleic acid, 

Fisswella barbadensis (limpet), 
sperm nucleic acid, 104 

Flagellum, see sperm-tail 

Flavin, in semen, 22, 33 

Flavonol pigments, in plant gam- 
etes, 72; as inhibitors of hyalur- 
onidase, 98 

Florence's reaction, 168 

Fluoride, effects on sperm and 
enzymes, 50-2, 56, 121, 156, 158 

Foetal fluids, fructose, 134 

Formaldehyde, effect on sperm, 57 

Forsythia, cross-pollination, 72 

Fowl semen {see also cock and 
turkey), 9, 11, 49, 59, 60, 65, 105 

Fox semen, 2, 44, 50 

Fractions in semen ejaculate, 35-8 

Freezing of sperm, 54, 63-5, 94 

Freezing point, and osmotic pres- 
sure of semen, 34-5 

Frequency of ejaculation, effect on 
semen, 34-5 

Frog sperm, irradiation effects, 59; 
pH and dilution effects, 60, 73; 
survival after freezing, 63-4; 
cytochrome, 90; nucleic acid, 105 

Fructolysis, 46-53, 151-9; index, 
46; relation to sperm density, 
motility and survival, 46-7, 151- 
3; after irradiation, 59; after 
temperature-shock, 63; inter- 
mediary enzymes and coenzymes, 
153-9; effect of adrenaline, 182; 
effect of thiol reagents and ergo- 
thioneine, 176-8 

Fructose, in semen and accessory 
organs, 16-24, 30-7, 135-8, 191; 
site and mechanism of formation, 
16, 121, 137-8, 149-151; utiliza- 
tion, 37, 46-7, 52-3, 150-9; in 

accessory gland transplants, 143- 
4; in gynogamone mucoprotein, 
71; in diluents, 65, 78, 79; rela- 
tion to quality of semen, 138, 
143, 193; relation to glucose, 
146-51; relation to inositol, 192; 
relation to androgens, 139^9; 
after castration, and hypophy- 
sectomy, 139-46; after scrotal 
application of heat, 62; after 
vasectomy, 138; in diabetes, 
146-8; relation to insulin, 146-8 
Fructosediphosphatase, 121 
Fructose test, for androgens, 68, 

139^6, 186 
Fucaceae, chemotropic substances, 

Fucose, in gynogamone mucopro- 
tein, 71 
Fumaric acid, and chemotaxis, 72 
Fungi, pigments in gametes, 72 

Gadushistone, 108 

Galactose, in gynogamone muco- 
protein, 71 

Galea capitis, 12-13 

Gamones, 69-73 

Gastrophilus intestinalis, fructose in 
haemolymph, 136 

Gelatin, in semen diluents, 80, 81; 
effect on sperm, 65 

Gelatin-liquefying enzyme, 96 

Gelation of semen, 28-31, 35, 184 

Gillichthys mirabilis (goby), protein 
in the vesicular secretion, 22 

Glandula vesicularis in rabbit, 21; 
fructose, 138; citric acid, 185 

Globulins, seminal, 112 

Glucose, in semen and accessory 
secretions, 21, 33, 137; utiliza- 
tion, 52-3, 150^-9; in gynogamone 
mucoprotein, 71; in diluents, 55- 
7, 65, 80-1; relation to fructose, 
121, 146-51, 157; in egg yolk 
and follicular fluid, 157; relation 
to inositol, 192 


The Biochemistry of Semen 

Glucose oxidase, use in semen 
analysis, 136 

Glucuronidase, in the prostatic 
secretion, 17 

Glutamic acid, in the prostate, 19, 
188; in mammalian semen, 84, 
113; in fish semen, 86; in semen 
diluents, 114; role in transamina- 
tions and citric acid formation, 

Glutamine, in the prostate, 20 

Glutathione, in semen and acces- 
sory glands, 20, 33, 176-7; 
effect on sperm, 58, 154, 176-7 

Glycerol, effect on sperm, 55; use 
in freezing, 64-6; in diluents, 81 

Glycerylphosphorylcholine, 16, 

Glycerylphosphorylcolamine, 126 

Glycine, in semen, 81, 85, 106, 
113-14; protection from light 
and dilution effect, 114; in 
diluents, 81, 114 

Glycogen, 5, 12, 129, 151-2 

Glycolysis, 46-8, 59, 151-4; in 
semen appraisal, 46-8; after 
irradiation, 59; in epididymal 
and ejaculated sperm, 1 53-4 

Goat, semen, fructose, 136; adrena- 
line, 181 

Goby, proteins of the vesicular 
secretion, 22 

Gomori's method, 13, 118 

Gonadectomy, effects, 113, 119, 
139-45, 186 

Gonadotrophins, 66, 68; influence 
on semen, 66, 68, 145-6, 148-9, 

Grasshopper, fructose in reproduc- 
tive organs, 136 

Guanine, 100-1 

Guinea-pig, accessory secretions 
and semen, 20, 23, 28, 126, 136, 
170, 172, 181-2; fertility, 6, 62 

Gum, in diluents, 81 

Gynogamones, 70-1 

Haematin, in seminal plasma, 22; 
in sperm, 88-93 

Haemocuprein, 89 

Haemospermia, 22 

Haliotis cracherodii (abalone), egg- 
membrane lysin, 95 

Hamster, fructose, 136; inositol 
deficiency, 191 

Heat, effect on sperm, 62 

Heavy metal compounds, effect on 
sperm, 57-8, 76, 98 

Hedgehog, copulatory plug, 28 

Heptacosane, 127-8 

Herring semen, nucleic acid, 99 
nuclear proteins, 99, 106-7: 
amino acids, 113; lipids, 124: 
agmatine and creatinine, 179 

Hesperidine, 98 

Hexokinase, 154-7 

Hexylresorcinol, effect on sperm, 

Histidine, 84, 108 

Histochemistry of sperm, 5, 10-13, 
118, 126 

Histological examination of semen, 

Histones, 98, 106-9 

Holothuria tubulosa, creatine and 
creatinine, 180 

Hormones {see also individual hor- 
mones), effect in vivo and in vitro, 
66-9, 139-45 

Human semen, see man, semen 

Hyaluronic acid, 93, 96 

Hyaluronidase, 93-8; release from 
sperm, 10, 83, 94-5; role, 96-8; 
inhibitors, 97-8; relation to 
citric acid, 1 88 

Hydrocarbons, chemotropic acti- 
vity, 72; in semen, 127-8 

Hydrogen ion concentration, in 
semen, 44—5; changes due to 
glycolysis, 44; relation to den- 
sity, motihty, and metabolism, 
44-5, 60-1; in accessory secre- 
tions, 17, 22 



Hydrogen peroxide, toxicity, 58, 92, 
93; formation by sperm, 58, 117, 
182; and X-ray action, 58; and 
oxygen damage, 58 

Hydrogen sulphide, effect on sperm, 

Hydroxylamine, effect on sperm, 91 

8-Hydroxyquinoline, as metal-bind- 
ing agent, 76 

Hyperpyrexia, relation to azoo- 
spermia, 62 

Hypertonicity, as sperm-affecting 
factor, 61-2 

Hypophysectomy, effect on semen, 
145-6, 186 

Hypophysis, role in semen forma- 
tion, 66, 145-9, 186 

Hypotonicity, as sperm-affecting 
factor, 61 

Immunology of semen, 79-80 

Impedance changes, frequency in 
semen, 41; relation to 'wave 
motion' and conception rate, 

India-ink staining method, appli- 
cation to sperm, 13 

Individual variations in the 
composition of semen, 32-5, 137, 
184, 191 

Indophenol reaction, 90-2 

Inhibitors of sperm motility and 
metabolism {see also spermicidal 
substances), 55-8, 91-2, 97-8 

Inner cylinder in the axial filament, 

Inosinic acid, dephosphorylation, 

Inositol, in semen and accessory 
secretions, 16, 21, 189-92; role, 

Insectivora, copulatory plug, 28; 
corpora amylacea, 132 

Insect sperm, transport, and sur- 
vival in the female tract, 9; PAS 
reaction, 12; activation by semi- 

nal plasma, 25; irradiation effects, 

59; fructose, 136 
Insulin, effect on seminal fructose, 

lodoacetamide and iodoacetate, 

effect on sperm, 56, 58, 158 
lodosobenzoate, effect on sperm 

56, 58, 177-8 
lodospermine, 168 
Ions and ionic equilibrium, 18, 23, 

25, 32-3, 42-4, 73-7, 188, 192 
Iron, protein-bound in sperm, 88- 

93; relation to haematin and 

cytochrome, 89; in karyogen, 109 
Irradiation effects, 58-60 
Isoagglutinin, 71 
Isoleucine, in mammalian semen, 

84, 113; in fish semen, 86, 106 

Jaffe's reaction, 1 79-80 

Jelly-coat of eggs, role in fertiliza- 
tion, 71; dissolving-factor, 71, 

Jelly fish, sperm nucleic acid, 104 

Karyogen, in salmon sperm, 109 

Keratin-like protein, in sperm- 
membrane, 110 

Key-hole limpet (Megathura crenu- 
lata), egg-membrane lysin, 95; 
nucleoprotein, 100 

Kinoplasmic droplet, 6-7, 154 

Lactic acid, content in semen and 
accessory secretions, 21, 32-3, 
36; as product of fructolysis, 46, 
152-8; as substrate for sperm, 
52-3, 153 

Lactic dehydrogenase, 158 

Lactose, and sperm metabohsm, 55, 

Lead salts, toxicity, 54 

Leakage of proteins from sperm, 
10, 57, 83, 94^5 

Lecithin, in semen and accessory 
secretions, 84, 124-5, 130-2 


The Biochemistry of Semen 

Leucine, in semen, 84, 113; in 
herring testes, 113; in diluents, 

Light effects, 66-7, 91, 114 

Light-reflection power of sperm, 8, 

Limpet, nucleic acid, 104 

Lipid, 124-33; redistribution dur- 
ing spermatogenesis, 6; in semen, 
32-3, 129-33; in sperm, 6, 86, 
124-7; in seminal plasma and 
accessory secretions, 130-3; meta- 
bolism, 49, 128-30, 172 

Lipid 'bodies', in semen, 131-2 

Lipid capsule, 6, 126; and deter- 
gents, 57; and senescence, 77, 83 

Lipoprotein, in sperm, 15, 125-6; 
loss due to damage and senes- 
cence, 77, 83, 84 

Lipositol, in L. G. B.-pabulum, 81; 
relation to inositol, 192 

Liquefaction, of semen, 28-9, 114; 
relation to citric acid, 188 

Lithobius forficatus, cytoplasmic 
lipids and steroids, 6 

Live-dead (differential) staining, 
39, 60, 63 

Locusta migratoria (grasshopper), 
fructose in reproductive organs, 

Longevity of sperm {see also 
survival), 9 

Lotahistone, 108 

Luminosity of sperm, 8, 41 

Lump-sucicer, cyclopterin, 106 

Lysine, in seminal proteins, 84, 108; 
free in human semen, 113; in 
herring testes, 113; in diluents, 114 

Lytechinus, dilution effect and 
amino acids, 76; sperm nucleic 
acid, 104 

Lytic agents, 92-8; relation to 
fertilization, 95-6 

Mackerel, scombrine, 106; scom- 
bron, 109 

Magnesium, in semen, 32-3; in 
prostatic calculi, 132; relation to 
motility, and metabolism, 77; 
effect on phosphatases, 121, 123 

Male accessory organs, see acces- 
sory organs 

Maleic acid, and chemotaxis in 
plants, 72 

Male sex hormone, see androgens, 
and testosterone 

Malic acid, role in chemotaxis, 71 

Malnutrition, effect on accessory 
organs and semen, 148-9 

Malonic acid, effect on sperm, 52 

Man, prostate, 16, 19, 181-88 

Man, prostatic secretion, physico- 
chemical properties, 17-18; con- 
tribution to semen, 35; enzymes, 
17, 114-15, 118-19, 170-1; elec- 
trolytes, 17-18; protein, 19; lipids 
and lipid bodies, 130-2; choles- 
terol, 130; spermine, 166-7; citric 
acid, 18, 183-8; zinc, 19 

Man, semen, volume and density, 
2; sperm structure, 11, 14-15; 
physico-chemical properties, 8, 
41-5, 60; composition, 32-3; 
fractions, 35; mineral constitu- 
tents, 18-19, 32-3, 89, 188; zinc 
and carbonic anhydrase, 19, 89; 
reducing substances and ascorbic 
acid, 33, 48, 179; fructose and 
fructolysis, 32-4, 48, 136, 147- 
48; coagulation and liquefaction, 
28, 188; proteins and proteose in 
seminal plasma, 111-12; mucoid 
substance, 112; amino acids, 
111-13; spermine, and spermi- 
dine, 160-8; choline and phos- 
phorylcholine, 130, 168-73; adren- 
aline, 181; sulphur and gluta- 
thione, 33; lipids and lipid bodies, 
101, 126, 130-2; cholesterol, 130; 
oestrogen and androgen content, 
68-9; heptacosane and fatty 
acids, 127-8; citric acid, 17, 



183-8; inositol, 191; nucleic 
acid, 102; Feulgen reaction, 101; 
PAS reaction, 112; nuclease, 123; 
phosphatases, 117-23; proteoly- 
tic enzymes, 114-16; cytochrome, 
66, 90-1; hyaluronidase, 94; 
amine oxidases, 167, 182; respira- 
tion, 41, 50, 90, 167; choline 
esterase, 173; hormonal aspects, 
143-8; freezing effect, 63-5; 
effect of arsenicals, 58 
Man, seminal vesicle, 16, 35; com- 
position of secretion, 20, 23^, 
137; monamine oxidase, 182; 
phosphorylcholine, 171; fructose, 
23^, 137 
Mannose, as substrate for sperm, 

52, 152, 154 
Manteau lipidique, 15, 126 
Marsupialia, copulatory plug, 

Maturation of sperm, see ripening 
Megathura cremdata (key-hole lim- 
pet), egg-membrane lysin, 95 
Mercury salts, toxicity, 54, 57 
Mesaconic acid, and chemotaxis, 72 
Metabolic regulator, 153-4 
Metabolism, chief characteristics, 
45; of carbohydrate, 45-53, 151- 
9; of lipid, 128-30; of protein, 
114-17; of nitrogenous bases, 1 67, 
169-73, 176-8, 180-2; of citric 
acid, 187-8; extraneous factors, 
55-9, 73, 79; role of adenosine 
triphosphate, 129, 155-9 
Metal-chelating agents, and sperm, 

76, 114 
Metalloproteins in semen, 88-93 
Methionine, in semen, 84, 87; as 
substrate for ergothioneine for- 
mation, 178 
Methoxyquinone, effect on sperm, 

5-Methylcytosine, 100-1 
Methylene-blue reduction test, 48, 

Methylhydroquinone, effect on 

sperm, 57 
Meyerhof oxidation quotient, 153 
Middle-piece, structure and com- 
position, 10, 13-14, 87; separa- 
tion from sperm-head and tail, 
86-87; indophenol reaction, 90-2; 
lipids, 126-30 
Milk, as diluent, 79, 81 
Milovanov's diluent, 80 
Milovanov's resistance test, 78 
Mitochondrial sheath, 6, 14, 126 
Mole, copulatory plug, 28 
Molluscs, lytic agents, 95; sperm 

nucleic acid, 104 
Monkey, prostatic phosphatase, 

Monoamine oxidase, 182 
Motility of sperm, types, 41-2; 
role of fibrils, 14; assessment and 
relation to fertility, 39-53; rela- 
tion to metabolism, 45-50, 91- 
2, 151-4; activators and inhibi- 
tors, 25, 55-8, 92; after irradia- 
tion, 59; dilution effect, 72-82; 
role of, ions, 74, 77, sulphydryl 
groups, 77, 176-8, gamones, 69- 
72, adenosine triphosphate, 154- 
9, spermine, 167, acetylcholine, 
173, ergothioneine, 176-8, citric 
acid, 188 
Motility test, for androgen, 67 
Mouse sperm, structure, 6, 7; irra- 
diation effects, 59; fructose, 136 
Mucopolysaccharase, 96 
Mucopolysaccharides, 12, 97 
Mucoprotein, in semen, 28, 33; in 

sea-urchin 'jelly coat', 71 
Mucus plug, in the honey-bee, 28 

Neck-piece (neck, sperm-neck), 13 
Necrospermy, effect on fructolysis, 

Niacin (nicotinic acid), 33 
Nicotinamide nucleotide, dephos- 

phorylation, 122 


The Biochemistry of Semen 

Niederland reaction, 18 

Nitrogen, total and non-protein, 
21,32-3,84-7, 111-13 

Nitrogen mustard, effect on sperm, 

Nitrogenous bases, in semen, 

Noradrenaline, in semen, 181-2 

Nucleases, 123 

Nucleic acid {see also deoxyri- 
bonucleic and ribonucleic acid), 
in developing and mature sperm 
cells, 5; detection and analysis, 5, 
86-8, 99-106; species charac- 
teristics, 101-6 

Nucleohistone, see nucleoproteins 

Nucleoprotamine, see nucleopro- 

Nucleoproteins, 5, 8, 11, 86, 98- 
100, 106-9 

5-Nucleotidase, 38, 121-2 

Nucleus, as part of sperm-head, 
7-12; removal from sperm, 85; 
constituents, 98-109 

Nutrition, effect on the com- 
position of semen, 148-9; nutrient 
requirements of spermatozoa, 
xiii-xiv, 46-53, 128-9, 151-9 

Odour of semen, relation to sper- 
mine and spermidine, 165-6 

Oestrogens, content in semen, 68; 
effect on phosphatases, 119; 
effect on seminal fructose, 144-5 

Opossum, clotting of semen, 28; 
fructose, 136 

Optical properties of semen, 40-1 

Osmotic properties of semen and 
accessory secretions, 7, 17-18, 
23, 43; freezing point depression, 
43; role of citric acid, 188; role of 
inositol, 192 

Outer cylinder in the axial filament, 

Ovarian androgens, 144-5, 186 

Ovulase, 95 

Ovulation, 9 

Oxaloacetic acid, conversion to 
citric acid, 188; as substrate, 52 

Oxygen damage, 58 

Oxygen tension, in vas deferens and 
epididymis, 61; effect on sperm 
motility, 73, 75, 151-2; effect on 
metabolism, 153 

Oxytocic, hormone, 67; properties 
of seminal plasma, 27 

Oyster sperm, glycogen, 6; respira- 
tion, 50, 52 

Pantothenic acid in semen, 33 

Paracentrotus {see also sea-urchin), 
sperm nucleic acid, 104; gelatin- 
liquefying enzyme, 96 

PAS reaction, 12, 112-13 

Pasteur effect, 153-4 

Penicillin, in diluents, 81 

Pepsinogen, 116 

Peptides, life-prolonging effect on 
sperm, 76; as products of pro- 
teolysis, 108, 111 

Perch, 106 

Percine, 106, 108 

Periodic acid-Schiff reaction, 12, 

Permeability of sperm, 10; changes 
due to senescence and damage, 

pH, see hydrogen ion concentration 

Pharmacological effects, of seminal 
plasma and accessory gland 
secretions, 26-8; of alkaloids, 56; 
of spermine, 162; of choline, 173; 
of adrenaline and noradrenaline, 

Phenolphthalein phosphate, as sub- 
strate for phosphatase, 1 1 8 

2-Phenoxyethanol, spermicidal ac- 
tion, 50 

Phenylalanine, in semen, 84, 113; 
oxidation, 117 

p-Phenylenediamine, oxidation, 



Phenylmercuric acetate, effect on 
sperm, 57 

Phenylphosphate, as substrate for 
phosphatase, 118 

Phosphatases, 1 17-23; see also acid 
and alkaline phosphatase, 5- 
nucleotidase, adenosinetriphos- 
phatases and pyrophosphatase 

Phosphate {see also phosphorus 
compounds), in semen and ac- 
cessory secretions, 18, 21, 32-3, 
107; in prostatic calculi, 132; in 
diluents, 77-8, 80-1; effect on 
sperm, 77; formation by phos- 
phatases, 117-23, 167, 171-2; 
esterification, 158 

Phosphoarginine, in sperm, 181 

Phosphocreatine, in sperm and 
testes, 180-1 

Phosphofructokinase, 157 

1-Phosphofructose, 120, 121, 

6-Phosphofructose, 118, 120, 121, 
150, 157 

Phosphoglucomutase, 150-1 

1-Phosphoglucose, 120, 150-1 

6-Phosphoglucose, 118, 120, 150-1, 

Phosphoglyceric acid, 158 

Phosphoglycerol, 117 

Phosphohexose isomerase, 121, 
150-1, 157 

Phospholipids, in mammalian se- 
men and accessory secretions, 
32-3, 107, 125-3; in fish sperm, 
124-5; metabolism, 127-30, 172 

6-Phosphomannose, 120 

Phosphopyruvic acid, 1 58 

Phosphorus compounds in semen 
and accessory secretions, 20-1, 
32-3, 36, 84-8, 99, 103, 107, 
129-30, 155-9, 167, 170-3; see 
also under individual compounds 

Phosphorylations, in lipid meta- 
bolism, 129; in carbohydrate 
metabolism, 10, 149-59 

Phosphorylcholine, in semen and 
accessory secretions, 16, 119, 168, 

Phosphorylethanolamine, in pros- 
tate, 20 

Phosphorylhesperidine, 98 

Phosphotriose, oxidation by sperm, 

Phosphotriose dehydrogenase, 158 

Physico-chemical properties of se- 
men, 40-5 

Picrocrocin, in Chlamydomonas, 72 

Pigments, 22, 70, 72, 90-2 

Pike, nucleic acid, 105; esocine, 106 

Pilocarpine, effect on prostate, 18 

Pituitary gland, role in semen for- 
mation, 66, 145-9 

Plants, chemotaxis and sperm-egg 
interacting substances, 71-2; 
sperm cytochrome, 92 

Plasmal, 101-2, 126-7 

Plasmalogens, 126-7 

Plasmin, 115 

Plasmolysis of spermatozoa, by 
enzymes, 14; by chemical agents, 
14, 85-6, 98 

Poisons, effect on sperm, 55-8; see 
also inhibitors and spermicidal 

Polymixin, in diluents, 81 

Porgy, sperm lipids, 124 

Post-sperm fraction, 35-8 

Potassium, in accessory secretions, 
18, 21-3; in mammalian semen, 
32-3, 36; in fish and sea-urchin 
semen, 43^; relation to citric 
acid, 44, 188; effect on sperm, 55, 

Preputial glands, 7-dehydrocholes- 
terol, 133 

Pre-sperm fraction, 33-8 

Progesterone, androgenic effect, 

Prohn, in semen, 85, 106, 113 

Propionic acid, as substrate, 52 

Prostaglandin, 26-7 


The Biochemistry of Semen 

Prostate gland, secretory function, 
16-20, 130-3, 168-73, 181, 184-8, 
zinc, 19; phosphatases, 117-19; 
corpora amylacea, 131-3; amino 
acids and transaminases, 19-20, 
188; in the female, 187 

Prostatic calculi, 131 

Prostatic cancer, 119 

Prostatic phosphatase, see acid 

Prostatic secretion, species charac- 
teristics, 16-20, 130, 184-5; pH, 
17; effect on sperm, 25; enzymes, 
17, 114-19; mineral constituents, 
18; fructose, 16, 138; citric acid, 
16-19, 183-8; lipids, 130-3; 
amino acids, 19, spermine, 166-7 

Protamines, 98-9, 106-9 

Proteins, extracellular, 19, 22, 76, 
84, 111-23; intracellular, 14, 43, 
83-110, 124; release from sperm, 
10, 57, 74, 83; in coagulation and 
liquefaction, 28-34, 114-15; effect 
on sperm, 55, 64, 76, 79 

Proteolytic enzymes, in semen and 
reproductive organs, 17-19, 29, 
96, 114-16; effect on sperm, 14 

Proteose, seminal, 19, 22, 111-14 

Psammechinus {see also sea-urchin), 

Pseudo-hypophysectomy, effects, 
148-9, 186 

Pteridium aquilinum, motility and 
cytochrome, 92 

Puranen reaction, 165 

Pyrophosphatase, 122 

Pyrophosphorylcholine, 171 

Pyruvic acid, metabolism, 50, 51, 
158; as substrate, 50-2, 158 

Quality of semen, relation to other 

properties of semen, metabolism 

and fertility, 38-53, 78, 92-3, 138 

Quercetin glycosides in pollen, 72 

Quinones, spermicidal properties, 

57, effect on hyaluronidase, 98 

Rabbit ampullar secretion, 138, 185 
Rabbit glandula seminalis, 21 
Rabbit glandula vesicularis, 16, 21, 

138, 172, 185 
Rabbit prostate, 16; fructose, 16, 
138, 144-5; adrenaline, 181; 
citric acid, 185 
Rabbit semen, volume and density, 
2, 31; sperm structure, 11; 
motility and fertility, in relation 
to extraneous factors, 6, 9, 56, 
59-62, 78, 97; physico-chemical 
properties, 41-^4, 60-2; gel, 28, 
34; fructose, 32, 34, 136-7, 140-7; 
citric acid, 184-6; lipids, 126, 
131; hyaluronidase, 94; respira- 
tion, 49-50; castration and hor- 
mone effects, 140-6 
Rabbit testis, choline, 170 
Radiation effects, 58-60 
Radioactive compounds, 178 
Ram semen, volume, density and 
composition, 2, 32-3, 107; 
sperm structure and disintegra- 
tion, 11, 87, 110, 126; time re- 
quired to reach the ovum, 9; 
nucleic acid, 55, 88, 102, 107; 
proteins, 88-110, 113; ammonia, 
32, 116; glycogen, 5; fructose and 
fructolysis, 32-4, 136, 152-9; 
enzymes, 87, 93, 120-2, 150-9, 
174; lipids, 107, 126; respiration 
and cytochrome, 49-53, 87, 90-1; 
phosphorus compounds, 107, 
155; citric acid, 184; temperature 
effect, 62; dilution effect, 77-9; 
irradiation effect, 59 
Rat coagulating gland, fructose 
formation, 16, 138-44; role in 
coagulation, 29 
Rat preputial gland, dehydrocho- 

lesterol, 133 
Rat prostate, structure and func- 
tion, 16, 19, 138, 184-5; in the 
female, 187; fructose, 19, 138-44; 
citric acid, 19, 185-7; zinc and 



carbonic anhydrase, 19, 20; 
amino acids and amines, 20; 
phosphatase, 120; transaminase, 
188; castration and hormone 
effects, 120, 141-9, 186-7; trans- 
plantation, 143-4 

Rat semen, sperm structure, 11; 
lipids, 6; copulatory plug, 28; 
choline, 1 69-70; irradiation effect, 

Rat seminal vesicle, 16, 187; com- 
position of secretion, 20, 172, 
185-7; hormone effects and trans- 
plantation, 120, 186 

Reducing substances, 20-4, 33, 48, 
135, 174-9; see also ascorbic 
acid, fructose, ergothioneine 

Refractive index of sperm, 8 

Reptiles, 94, 105 

Resistance to dilution, 78 

Respiration of sperm, 49-53, 91-2, 
117, 129-31, 152-4, 167; sub- 
strates, 50-3 ; inhibitors, 49-51, 
56; after irradiation, 59; pH- 
optimum, 60; after temperature- 
shock, 63; in presence of 'egg- 
water', 76 

Rhinolophidae, copulatory plug, 

Riboflavin, 22, 33 

Ribonucleic acid, 5, 86 

Ring centriole, 14 

Ringer solution, as semen diluent, 

Ripening of sperm, 4-8, 25, 41-3, 
90, 153-4, 178 

Rodents {see also guinea-pig, rab- 
bit, rat), clotting phenomenon in 
semen, 28 

Rutin, in pollen, 72 

Salmine, 106, 108 

Salmofario {see also trout), nucleo- 

protein and nucleic acid, 99, 105 
Salmo fontinalis, constituents of 

seminal plasma, 84 

Salmon semen, mineral consti- 
tuents, 43, 84; proteins, 84-6, 
98-9, 106-9, 125; nucleic acid, 
86, 103; lipids and cholesterol, 86, 
124-5; free amino acids, 86; cata- 
lase, 92; phosphatases, 86; andro- 
gamones, 71 

Salt effects on sperm, 55, 62 

Sand-dollar, cyanide effect on 
sperm, 92 

Saponin, effect on sperm, 57 

Saxostrea commercialis (oyster), 
respiration of sperm, 50 

Scherer-Salkowski reaction, 190 

Schiff's reagent, 11, 126 

Scombrine, 106 

Scombrone, 109 

Scylliorhinus caniculus (dogfish), 
fructose, 136 

Sea-cucumber, sperm nucleic acid, 

Sea-urchin semen (see also Arbacia, 
Echinus, Lytechinus, Paracentro- 
tus, Psammechinus, Strongylocen- 
trotus), density, 74; pH, 44; potas- 
sium, 44; glycogen, 6, 129; nucleic 
acid, 100, 104; nuclease, 123; 
proteins, 84, 108; proteolytic 
enzymes and 'lytic agents', 95-6; 
sperm-egg interacting substances, 
69-71, 95-6; lipids and sterols, 
125, 129; adenosine triphosphate, 
155; creatine and creatinine, 180; 
cytochrome, 90-2; catalase, 93; 
copper, 90; effects of radiation, 
oxygen, dilution and other ex- 
traneous factors, 52-9, 74-7, 
91-2, 114; metabolism, 50, 52, 
56, 74, 76, 91-2, 129-31, 151 

Semen composition, tables, 2, 20, 
21, 32-6, 84-90, 103-7, 130, 169, 
185, 191 

Seminal duct, 7, 15, 61, 138 

Seminal plasma, origin, composi- 
tion and function, 15-29; separa- 
tion from sperm, 83; proteins, 


The Biochemistry of Semen 

Seminal plasma {contd.) — 
amino acids and enzymes, lll- 
23; lipids, 130-3; fructose, 135-59; 
nitrogenous bases, 160-82; citric 
acid, 183-8; inositol, 189-92 

Seminal stains, detection, 163-8 

Seminal vesicles, secretory func- 
tion, 15-24, 35-8, 191; amino 
acids, 113; phosphatases, 120-2; 
lipid, 131-3; fructose, 137-8, 
142; choline, 167-73; ergothio- 
neine, 175-6; citric acid, 184-8; 
inositol, 190-2; role in coagula- 
tion, 29 

Seminiferous epithelium {see also 
spermatogenesis), 4, 59, 94 

Senescence, 12, 73-9, 83 

Serine, 85, 106, 113 

Sertoli cells, glycogen, 5; lipids, 6 

Snail, sperm nucleic acid, 104 

Sodium, 18, 21, 32-3, 36, 43, 188 

Specific gravity, 7-8, 42-3 

Spermatheca, 9 

Spermatic veil ('floating cap'), 12 

Spermatids, origin, 1-4; glycogen, 5 

Spermatin (spermine), 161 

Spermatocytes, basophilic cyto- 
plasm, 1-4; glycogen, 5; lipid, 6 

Spermatogenesis, accompanying 
chemical phenomena, 4-8, 109; 
onset, 62, 140-1, 186; length, 
178-9; dependence on hormones, 
nutrition and other factors, 62, 
66-7, 72, 143, 148-9; after 
irradiation, 59 

Spermatogonia, 4-6 

Spermatophore, 9 

Spermatozoa, 1-15, 38-45; separa- 
tion and disintegration, 83-8; 
metabolism, 45-54, 117, 128-33, 
151-9, 177-8; nucleic acid and 
proteins, 83-123; lipids, 125-30 

Spermatozoids in mosses and ferns, 

Sperm-density, species characteris- 
tics, 1-2, 31-6, 82; relation to 

other properties and metabolism 
of semen, 39-53, 138, 193 

Sperm-egg interacting substances, 

Sperm-head, 10-13, 85-6, 94, 98- 
110, 126 

Spermicidal substances, 50, 54-8, 
79-80, 97-8 

Spermidine, 166-7 

Spermine, 2, 160-8 

Sperm-lysin, 71, 95-6 

Sperm-maturation, see ripening 

Sperm-membrane, 13, 110 

Sperm middle-piece,5eemiddle-piece 

Sperm-rich fraction, 35-8 

Sperm-ripening, see ripening 

Sperm-tail, 10, 14-15, 85-7, 124-6 

Spermosin, 14 

Sphingomyelin, 124 

Spiral body, 14, 126 

Split-ejaculate method, use in 
semen analysis, 35-8 

Staining methods, 12-13, 39, 60, 63, 
100-1, 126 

Stallion reproductive organs, cho- 
line, 168; citric acid, 184 

Stallion semen, volume and dens- 
ity, 2; abnormalities, 11; physico- 
chemical properties, 41-4; frac- 
tions, 38; gelation, 28; composi- 
tion, 32-3; androgen content, 69; 
citric acid, 184; fructose, 32, 136; 
choline, 170; creatine and creati- 
nine, 180; freezing and dilution 
effects, 63, 78 

Starfish sperm, 92, 114 

Sterility, see fertility 

Steroids in semen and reproductive 
organs, 6, 67-9, 86, 124-5, 130-3; 
urinary excretion, 62 

Stichopus diabole (sea-cucumber), 
sperm nucleic acid, 104 

Stilboestrol, 144-5 

Storage of sperm, in the epididymis, 
6-7; in the female tract, 8-9; with 
diluents, 79-82 



Streptomycin, in diluents, 81 

Strongylocentrotiis, (see also sea- 
urchin), 114, 180 

Sturgeon sperm, 104, 106, 108 

Sturine, 106, 108 

Succinic acid, oxidation, 50, 52, 57, 
87, 92 

Succinic dehydrogenase, 87, 92 

Sucrose, effect on sperm, 55, 152; 
use in freezing, 64, 65; in 
chemotaxis, 71 

Sulphate, 21, 77, 175-8 

Sulphite, 179 

Sulphonamides, excretion in semen, 
57; in diluents, 81 

Sulphur, elementary, 154; com- 
pounds, 21-3, 33, 56-8, 71, 77, 
84-7, 110, 154, 176-9 

Sulphydryl-binding substances, 56- 
8, 176-8 

Sulphydryl groups, function, 23, 
56-8, 77, 154, 176-9 

Surface-active substances, spermici- 
dal properties, 50, 57 

Survival of sperm, 6-9, 24-5, 60-1, 
66-7, 78-82, 151-4 

Tail-sheath (see also sperm-tail), 

Taurine, in prostate, 20 

Temperature, effects on sperm, 12, 

Temperature shock, 12; preven- 
tion, 80 

Tench, nucleic acid, 105 

Testis, role in sperm formation, 
4-6; spermine, 162; choline, 170; 
creatinine, 179; cytochrome, 90; 
hyaluronidase, 94; phosphocrea- 
tine, 180-1 

Testosterone, 69; effect on the 
composition of semen, 113, 139- 
45, 185-7 

Thiamine, 33 

Thioglycolic acid, effect on sperm- 
membrane, 110 

Thiol-groups, function, 23, 56-8, 
77, 154, 176-9 

Thiolhistidine, relation to ergo- 
thioneine, 174, 178 

Thiol-reagents, 56-8, 176-8 

Threonine, 84, 113 

Thymine, 100, 101 

Thynnine, 106 

Thyroxine, effect on sperm, 68, 71 

Toluquinone, effect on sperm, 57 

Tonicity, as sperm-affecting factor, 

Transaminase, 19, 186 

Transferase activity of phospha- 
tase, 119 

Transmethylations, 172, 178 

Transplants of accessory organs, 
secretory function, 143-4, 186 

Transport of spermatozoa, in the 
male and female, 6-9, 178-9 

ethanol, effect on sperm, 57 

Trimethylbenzylammonium chlor- 
ide, effect on sperm-membrane, 

Triphenyltetrazolium chloride, 
effect on sperm, 56 

Trithiolpropane, effect on sperm, 

Trout semen, nucleic acid and 
nuclear proteins, 99-109; potas- 
sium, 43, 74; effects of oxygen, 
dilution and ions, 73-4 

Tryptic enzyme, 1 1 5 

Tryptophan, in semen, 84, 109; 
oxidation, 117 

Tunny fish, thynnine, 106 

Turbidity of semen, 40-1 

Turkey semen, volume and density, 

Turpentine, effect on sperm, 54 

Tyrosine, in semen, 85, 86, 108, 113; 
oxidation, 117 

Ultrasonic waves, use for sperm 
disintegration, 86, 100 


The Biochemistry of Semen 

Urea, in semen, 32, 116; effect on 
sperm, 55 

Urechis caupo, creatine and creati- 
nine, 180 

Urethral glands, 15, 37-8 

Uric acid in semen, 32, 123 

Vitamins, role in reproduction 
and semen formation, 23, 72, 

Vitrification, 64-6 

Volume of semen, 1-2, 31-6 

Washing, effect on sperm, 79-84 
Wave motion in semen, 41 
Witte's peptone, in semen diluent, 

Wolffian duct, 21 

Xanthine oxidase, 22, 123 
X-rays, effect on sperm, 58-9 

Zinc, in semen and prostate, 19, 

Zymohexase, 157